Methods for increasing product yields

ABSTRACT

A non-naturally occurring microbial organism includes a microbial organism having a reductive TCA or Wood-Ljungdahl pathway in which at least one exogenous nucleic acid encoding these pathway enzymes is expressed in a sufficient amount to enhance carbon flux through acetyl-CoA. A method for enhancing carbon flux through acetyl-CoA includes culturing theses non-naturally occurring microbial organisms under conditions and for a sufficient period of time to produce a product having acetyl-CoA as a building block. Another non-naturally occurring microbial organism includes at least one exogenous nucleic acid encoding an enzyme expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of carbon monoxide or hydrogen, thereby increasing the yield of redox-limited products via carbohydrate-based carbon feedstock. A method for enhancing the availability of reducing equivalents in the presence of carbon monoxide or hydrogen includes culturing this organism for a sufficient period of time to produce a product.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/806,435 filedJul. 22, 2015 (now U.S. Pat. No. 10,087,470), which is a continuation ofU.S. Ser. No. 13/889,056 filed May 7, 2013, which is a continuation ofU.S. Ser. No. 13/011,788 filed Jan. 21, 2011 (now U.S. Pat. No.8,445,244), which claims the benefit of priority under 35 U.S.C § 119(e)to U.S. Provisional Application 61/307,437, filed Feb. 23, 2010 and U.S.Provisional Application 61/314,570, filed Mar. 16, 2010, each of whichis incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

This invention relates generally to biosynthetic processes and, morespecifically to organisms having enhanced carbon fixation capabilities.

1,3-butanediol (1,3-BDO) is a four carbon diol traditionally producedfrom acetylene via its hydration. The resulting acetaldehyde is thenconverted to 3-hydroxybutyraldehdye which is subsequently reduced toform 1,3-BDO. More recently, acetylene has been replaced by the lessexpensive ethylene as a source of acetaldehyde. 1,3-BDO is commonly usedas an organic solvent for food flavoring agents. It is also used as aco-monomer for polyurethane and polyester resins and is widely employedas a hypoglycemic agent. Optically active 1,3-BDO is a useful startingmaterial for the synthesis of biologically active compounds and liquidcrystals. Another use of 1,3-butanediol is that its dehydration affords1,3-butadiene (Ichikawa et al. Journal of Molecular Catalysis A-Chemical256:106-112 (2006); Ichikawa et al. Journal of Molecular CatalysisA-Chemical 231:181-189 (2005), which is useful in the manufacturesynthetic rubbers (e.g., tires), latex, and resins. The reliance onpetroleum based feedstocks for either acetylene or ethylene warrants thedevelopment of a renewable feedstock based route to 1,3-butanediol andto butadiene.

Isopropanol (IPA) is a colorless, flammable liquid that mixes completelywith most solvents, including water. The largest use for IPA is as asolvent, including its well known yet small use as “rubbing alcohol,”which is a mixture of IPA and water. As a solvent, IPA is found in manyeveryday products such as paints, lacquers, thinners, inks, adhesives,general-purpose cleaners, disinfectants, cosmetics, toiletries,de-icers, and pharmaceuticals. Low-grade IPA is also used in motor oils.IPA is also used as a chemical intermediate for the production ofisopropylamines (Ag products), isopropylethers, and isopropyl esters.Isopropanol is manufactured by two petrochemical routes. The predominantprocess entails the hydration of propylene either with or withoutsulfuric acid catalysis. Secondarily, IPA is produced via hydrogenationof acetone, which is a by-product formed in the production of phenol andpropylene oxide.

4-hydroxybutanoic acid (4-hydroxybutanoate, 4-hydroxybutyrate, 4-HB) isa 4-carbon carboxylic acid that is used as a building block for variouscommodity and specialty chemicals. In particular, 4-HB can serve as anentry point into the 1,4-butanediol family of chemicals, which includessolvents, resins, polymer precursors, and specialty chemicals.

1,4-butanediol (BDO) is a valuable chemical for the production of highperformance polymers, solvents, and fine chemicals. It is the basis forproducing other high value chemicals such as tetrahydrofuran (THF) andgamma-butyrolactone (GBL). The value chain is comprised of three mainsegments including: (1) polymers, (2) THF derivatives, and (3) GBLderivatives. In the case of polymers, BDO is a comonomer forpolybutylene terephthalate (PBT) production. PBT is a medium performanceengineering thermoplastic used in automotive, electrical, water systems,and small appliance applications. Conversion to THF, and subsequently topolytetramethylene ether glycol (PTMEG), provides an intermediate usedto manufacture spandex products such as LYCRA® fibers. PTMEG is alsocombined with BDO in the production of specialty polyester ethers(COPE). COPEs are high modulus elastomers with excellent mechanicalproperties and oil/environmental resistance, allowing them to operate athigh and low temperature extremes. PTMEG and BDO also make thermoplasticpolyurethanes processed on standard thermoplastic extrusion,calendaring, and molding equipment, and are characterized by theiroutstanding toughness and abrasion resistance. The GBL produced from BDOprovides the feedstock for making pyrrolidones, as well as serving theagrochemical market. The pyrrolidones are used as high performancesolvents for extraction processes of increasing use, including forexample, in the electronics industry and in pharmaceutical production.

BDO is produced by two main petrochemical routes with a few additionalroutes also in commercial operation. One route involves reactingacetylene with formaldehyde, followed by hydrogenation. More recentlyBDO processes involving butane or butadiene oxidation to maleicanhydride, followed by hydrogenation have been introduced. BDO is usedalmost exclusively as an intermediate to synthesize other chemicals andpolymers.

Thus, there exists a need for the development of methods for effectivelyproducing commercial quantities of compounds such as 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol. The present inventionsatisfies this need and provides related advantages as well. Additionalproduct molecules that can be produced by the teachings of thisinvention include but are not limited to ethanol, butanol, isobutanol,isopropanol, succinic acid, fumaric acid, malic acid, 3-hydroxypropionicacid, lactic acid, adipic acid, 6-aminocaproic acid,hexamethylenediamine, caprolactam, 3-hydoxyisobutyric acid,2-hydroxyisobutyric acid, methacrylic acid, acrylic acid,1,3-propanediol, glycerol, and long chain hydrocarbons, alcohols, acids,and esters.

SUMMARY OF THE INVENTION

In some aspects, embodiments disclosed herein relate to a non-naturallyoccurring microbial organism that includes a microbial organism having areductive TCA pathway which includes at least one exogenous nucleic acidencoding a reductive TCA pathway enzyme expressed in a sufficient amountto enhance carbon flux through acetyl-CoA. The at least one exogenousnucleic acid is selected from an ATP-citrate lyase, citrate lyase, afumarate reductase, and an alpha-ketoglutarate:ferredoxinoxidoreductase. In some aspects, embodiments disclosed herein relate toa method for enhancing carbon flux through acetyl-CoA that includesculturing this non-naturally occurring microbial organism underconditions and for a sufficient period of time to produce a producthaving acetyl-CoA as a building block.

In some aspects, embodiments disclosed herein relate to a non-naturallyoccurring microbial organism that includes a microbial organism having aWood-Ljungdahl pathway which includes at least one exogenous nucleicacid encoding a Wood-Ljungdahl pathway enzyme expressed in a sufficientamount to enhance carbon flux through acetyl-CoA. The at least oneexogenous nucleic acid is selected from a) Formate dehydrogenase, b)Formyltetrahydrofolate synthetase, c) Methenyltetrahydrofolatecyclohydrolase, d) Methylenetetrahydrofolate dehydrogenase, e)Methylenetetrahydrofolate reductase, f) Methyltetrahydrofolate:corrinoidprotein methyltransferase (AcsE), g) Corrinoid iron-sulfer protein(AcsD), h) Nickel-protein assembly protein (AcsF & CooC), i) Ferredoxin(Orf7), j) Acetyl-CoA synthase (AcsB & AcsC), k) Carbon monoxidedehydrogenase (AcsA), and l) Pyruvate ferredoxin oxidoreductase orpyruvate dehydrogenase, and m) pyruvate formate lyase. In some aspects,embodiments disclosed herein relate to a method for enhancing carbonflux through acetyl-CoA that includes culturing this non-naturallyoccurring microbial organism under conditions and for a sufficientperiod of time to produce a product having acetyl-CoA as a buildingblock.

In some aspects, embodiments disclosed herein relate to a non-naturallyoccurring microbial organism that includes a microbial organism having amethanol Wood-Ljungdahl pathway which includes at least one exogenousnucleic acid encoding a methanol Wood-Ljungdahl pathway enzyme expressedin a sufficient amount to enhance carbon flux through acetyl-CoA. The atleast one exogenous nucleic acid is selected from a) Methanolmethyltransferase (MtaB), b) Corrinoid protein (MtaC), c)Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), d)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), e)Corrinoid iron-sulfer protein (AcsD), f) Nickel-protein assembly protein(AcsF & CooC), g) Ferredoxin (Orf7), h) Acetyl-CoA synthase (AcsB &AcsC), i) Carbon monoxide dehydrogenase (AcsA), j) Pyruvate ferredoxinoxidoreductase or pyruvate dehydrogenase, k) pyruvate formate lyase, andl) NAD(P)H:ferredoxin oxidoreductase. In some aspects, embodimentsdisclosed herein relate to a method for enhancing carbon flux throughacetyl-CoA, comprising culturing this non-naturally occurring microbialorganism under conditions and for a sufficient period of time to producea product having acetyl-CoA as a building block.

In some aspects, embodiments disclosed herein relate to a non-naturallyoccurring microbial organism that includes at least one exogenousnucleic acid encoding an enzyme expressed in a sufficient amount toenhance the availability of reducing equivalents in the presence ofcarbon monoxide or hydrogen, thereby increasing the yield ofredox-limited products via carbohydrate-based carbon feedstock. The atleast one exogenous nucleic acid is selected from a carbon monoxidedehydrogenase, a hydrogenase, an NAD(P)H:ferredoxin oxidoreductase, anda ferredoxin. In some aspects, embodiments disclosed herein relate to amethod for enhancing the availability of reducing equivalents in thepresence of carbon monoxide or hydrogen thereby increasing the yield ofredox-limited products via carbohydrate-based carbon feedstock, themethod includes culturing this non-naturally occurring microbialorganism under conditions and for a sufficient period of time to producea product.

In some aspects, embodiments disclosed herein relate to a non-naturallyoccurring microbial organism that includes a microbial organism having areductive TCA pathway which includes at least one exogenous nucleic acidencoding a reductive TCA pathway enzyme. the at least one exogenousnucleic acid is selected from an ATP-citrate lyase, citrate lyase, afumarate reductase, and an alpha-ketoglutarate:ferredoxinoxidoreductase; and at least one exogenous enzyme selected from a carbonmonoxide dehydrogenase, a hydrogenase, a NAD(P)H:ferredoxinoxidoreductase, and a ferredoxin, expressed in a sufficient amount toallow the utilization of 1) CO, 2) CO₂ and H₂, 3) CO and CO₂, 4)synthesis gas comprising CO and H₂, and 5) synthesis gas comprising CO,CO₂, and H₂.

In some aspects, embodiments disclosed herein relate to a method thatincludes culturing a non-naturally occurring microbial organism thatincludes a microbial organism having a reductive TCA pathway comprisingat least one exogenous nucleic acid encoding a reductive TCA pathwayenzyme. The at least one exogenous nucleic acid is selected from anATP-citrate lyase, citrate lyase, a fumarate reductase, and analpha-ketoglutarate:ferredoxin oxidoreductase; and at least oneexogenous enzyme selected from a carbon monoxide dehydrogenase, ahydrogenase, a NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin,expressed in a sufficient amount to allow the utilization of 1) CO, 2)CO₂ and H₂, 3) CO and CO₂, 4) synthesis gas comprising CO and H₂, and 5)synthesis gas comprising CO, CO₂, and H₂ to produce a product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a shows the pathways for the biosynthesis of 1,3-butanediol fromacetyl-CoA; the enzymatic transformations shown are carried out by thefollowing enzymes: 1) Acetoacetyl-CoA thiolase (AtoB), 2)Acetoacetyl-CoA reductase (CoA-dependent, alcohol forming), 3)3-oxobutyraldehyde reductase (aldehyde reducing), 4)4-hydroxy,2-butanone reductase, 5) Acetoacetyl-CoA reductase(CoA-dependent, aldehyde forming), 6) 3-oxobutyraldehyde reductase(ketone reducing), 7) 3-hydroxybutyraldehyde reductase, 8)Acetoacetyl-CoA reductase (ketone reducing), 9) 3-hydroxybutyryl-CoAreductase (aldehyde forming), 10) 3-hydroxybutyryl-CoA reductase(alcohol forming), 11) an acetoacetyl-CoA transferase, anacetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or aphosphotransacetoacetylase/acetoacetate kinase, 12) Acetoacetatereductase, 13) 3-hydroxybutyryl-CoA transferase, hydrolase, orsynthetase, 14) 3-hydroxybutyrate reductase, and 15) 3-hydroxybutyratedehydrogenase.

FIG. 1b shows the pathways for the biosynthesis of isopropanol fromacetyl-CoA; the enzymatic transformations shown are carried out by thefollowing enzymes: 1) Acetoacetyl-CoA thiolase (AtoB), 2)Acetoacetyl-CoA transferase, acetoacetyl-CoA hydrolase, acetoacetyl-CoAsynthetase, or phosphotransacetoacetylase/acetoacetate kinase, 3)Acetoacetate decarboxylase (Adc), and 4) Isopropanol dehydrogenase (Adh)

FIG. 1c shows the pathways for the biosynthesis of 4-hydroxybutyrate(4-HB); the enzymatic transformations shown are carried out by thefollowing enzymes: 1) Acetoacetyl-CoA thiolase (AtoB), 2)3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3) Crotonase (Crt), 4)Crotonyl-CoA hydratase (4-Budh), 5) 4-Hydroxybutyryl-CoA transferase,hydrolase or synthetase, 6) Phosphotrans-4-hydroxybutyrylase, and 7)4-Hydroxybutyrate kinase.

FIG. 1d shows the pathways for the biosynthesis of 1,4-butanediol; theenzymatic transformations shown are carried out by the followingenzymes: 1) Acetoacetyl-CoA thiolase (AtoB), 2) 3-Hydroxybutyryl-CoAdehydrogenase (Hbd), 3) Crotonase (Crt), 4) Crotonyl-CoA hydratase(4-Budh), 5) 4-hydroxybutyryl-CoA reductase (alcohol forming), 6)4-hydroxybutyryl-CoA reductase (aldehyde forming), 7) 1,4-butanedioldehydrogenase, 8) 4-Hydroxybutyryl-CoA transferase, 4-Hydroxybutyryl-CoAsynthetase, 4-Hydroxybutyryl-CoA hydrolase, orPhosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate kinase, and 9)4-Hydroxybutyrate reductase.

FIG. 2a shows the reverse TCA cycle for fixation of CO₂ on carbohydratesas substrates. The enzymatic transformations are carried out by theenzymes as shown.

FIG. 2b shows the flux distribution showing an enhanced maximumtheoretical yield of isopropanol on glucose when carbon is routed viathe reductive TCA cycle

FIG. 3a shows a flow diagram depicting the Wood-Ljungdahl pathway andformation routes for acetate and ethanol; the transformations that arecarried out in organisms capable of growth on synthesis gas are 1) COdehydrogenase, 2) hydrogenase, 3) energy-conserving hydrogenase (ECH),and 4) bi-functional CO dehydrogenase/acetyl-CoA synthase.

FIG. 3b shows a pathway for the utilization of methanol for theformation of acetyl-CoA; the enzymatic transformations shown are carriedout by the following enzymes: 1) Methanol methyltransferase (MtaB), 2)Corrinoid protein (MtaC), 3) Methyltetrahydrofolate:corrinoid proteinmethyltransferase (MtaA), 4) Methyltetrahydrofolate:corrinoid proteinmethyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6)Nickel-protein assembly protein (AcsF & CooC), 7) Ferredoxin (Orf7), 8)Acetyl-CoA synthase (AcsB & AcsC), and 9) Carbon monoxide dehydrogenase(AcsA).

FIG. 4a shows a pathway for enabling carbon fixation from syngas intoacetyl CoA. The reducing equivalents are derived from carbohydrates suchas glucose. The enzymatic transformations are carried out by thefollowing enzymes: 1) Formate dehydrogenase, 2) Formyltetrahydrofolatesynthetase, 3) Methenyltetrahydrofolate cyclohydrolase, 4)Methylenetetrahydrofolate dehydrogenase, 5) Methylenetetrahydrofolatereductase, 6) Methyltetrahydrofolate:corrinoid protein methyltransferase(AcsE), 7) Corrinoid iron-sulfur protein (AcsD), 8) Nickel-proteinassembly protein (AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoAsynthase (AcsB & AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12)Pyruvate formate lyase (Pfl), 13) Pyruvate ferredoxin oxidoreductase(Por) or pyruvate dehydrogenase (PDH).

FIG. 4b shows a pathway for enabling carbon fixation from methanol intoacetyl CoA. The reducing equivalents are derived from carbohydrates suchas glucose. “n” depicts the number of moles of methanol that areprovided. The enzymatic transformations are carried out by the followingenzymes: 1) Methanol methyltransferase (MtaB), 2) Corrinoid protein(MtaC), 3) Methyltetrahydrofolate:corrinoid protein methyltransferase(MtaA), 4) Methyltetrahydrofolate:corrinoid protein methyltransferase(AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Carbon monoxidedehydrogenase (AcsA), 7) Nickel-protein assembly protein (AcsF & CooC),8) Ferredoxin (Orf7), 9) Acetyl-CoA synthase (AcsB & AcsC), 10) Pyruvateferredoxin oxidoreductase (Por), 11) Pyruvate dehydrogenase (PDH), 12)Pyruvate formate lyase (Pfl), 13) Formate dehydrogenase

FIG. 5a shows the flux distribution with an enhanced maximum theoreticalyield of 1,3-butanediol on glucose when carbon fixation via theWood-Ljungdahl pathway is employed in the absence of methanol; theenzymatic transformations shown are carried out by the followingenzymes: 1) Formate dehydrogenase, 2) Formyltetrahydrofolate synthetase,3) Methenyltetra-hydrofolate cyclohydrolase, 4)Methylenetetrahydrofolate dehydrogenase, 5) Methylenetetra-hydrofolatereductase, 6) Methyltetrahydrofolate:corrinoid protein methyltransferase(AcsE), 7) Corrinoid iron-sulfur protein (AcsD), 8) Nickel-proteinassembly protein (AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoAsynthase (AcsB & AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12)Pyruvate formate lyase (Pfl), 13) Pyruvate ferredoxin oxidoreductase(Por) or pyruvate dehydrogenase (PDH), 14) Acetoacetyl-CoA thiolase(AtoB), 15) Acetoacetyl-CoA reductase (CoA-dependent, alcohol forming),16) 3-Oxobutyraldehyde reductase (aldehyde reducing), 17)4-Hydroxy-2-butanone reductase, 18) Acetoacetyl-CoA reductase(CoA-dependent, aldehyde forming), 19) 3-Oxobutyraldehyde reductase(ketone reducing), 20) 3-Hydroxybutyraldehyde reductase, 21)Acetoacetyl-CoA reductase (ketone reducing), 22) 3-Hydroxybutyryl-CoAreductase (aldehyde forming), 23) 3-Hydroxybutyryl-CoA reductase(alcohol forming); when glucose is fed in the presence of theWood-Ljungdahl pathway, a yield increase from 1 mol 1,3-butanediol/molglucose to 1.09 mol 1,3-butanediol/mol glucose is realized.

FIG. 5b shows the flux distribution with an enhanced maximum theoreticalyield of 1,3-butanediol from glucose when carbon fixation via themethanol Wood-Ljungdahl pathway is employed using both syngas andmethanol; the enzymatic transformations shown are carried out by thefollowing enzymes: 1) Methanol methyltransferase (MtaB), 2) Corrinoidprotein (MtaC), 3) Methyltetrahydrofolate:corrinoid proteinmethyltransferase (MtaA), 4) Methyltetrahydro-folate: corrinoid proteinmethyltransferase (AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6)Carbon monoxide dehydrogenase (AcsA), 7) Nickel-protein assembly protein(AcsF & CooC), 8) Ferredoxin (Orf7), 9) Acetyl-CoA synthase (AcsB &AcsC), 10) Pyruvate ferredoxin oxidoreductase (Por), 11) Pyruvatedehydrogenase (PDH), 12) Pyruvate formate lyase (Pfl), 13) Formatedehydrogenase, 14) Acetoacetyl-CoA thiolase (AtoB), 15) Acetoacetyl-CoAreductase (CoA-dependent, alcohol forming), 16) 3-Oxobutyraldehydereductase (aldehyde reducing), 17) 4-Hydroxy-2-butanone reductase, 18)Acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), 19)3-Oxobutyraldehyde reductase (ketone reducing), 20)3-Hydroxybutyraldehyde reductase, 21) Acetoacetyl-CoA reductase (ketonereducing), 22) 3-Hydroxybutyryl-CoA reductase (aldehyde forming), 23)3-Hydroxybutyryl-CoA reductase (alcohol forming); when glucose andmethanol are fed in 1.0:0.4 ratio, it affords an increase from 1 mol1,3-butanediol/mol glucose to 1.2 mol 1,3-butanediol/mol glucose.

FIG. 6a shows the flux distribution with an enhanced maximum theoreticalyield of isopropanol from glucose when carbon fixation via theWood-Ljungdahl pathway is employed in the absence of methanol; theenzymatic transformations shown are carried out by the followingenzymes: 1) Formate dehydrogenase, 2) Formyltetrahydrofolate synthetase,3) Methenyltetra-hydrofolate cyclohydrolase, 4)Methylenetetrahydrofolate dehydrogenase, 5) Methylenetetrahydrofolatereductase, 6) Methyltetrahydrofolate:corrinoid protein methyltransferase(AcsE), 7) Corrinoid iron-sulfur protein (AcsD), 8) Nickel-proteinassembly protein (AcsF & CooC), 9) Ferredoxin (Orf7), 10) Acetyl-CoAsynthase (AcsB & AcsC), 11) Carbon monoxide dehydrogenase (AcsA), 12)Pyruvate formate lyase (Pfl), 13) Pyruvate ferredoxin oxidoreductase(Por) or pyruvate dehydrogenase (PDH), 14) Acetoacetyl-CoA thiolase(AtoB), 15) Acetoacetyl-CoA:acetate:CoA transferase (AtoAD), 16)Acetoacetate decarboxylase (Adc), and 17) Isopropanol dehydrogenase(Adh).

FIG. 6b shows the flux distribution with an enhanced maximum theoreticalyield of isopropanol from glucose when carbon fixation via the methanolWood-Ljungdahl pathway is employed using both syngas and methanol; theenzymatic transformations shown are carried out by the followingenzymes: 1) Methanol methyltransferase (MtaB), 2) Corrinoid protein(MtaC), 3) Methyltetrahydrofolate:corrinoid protein methyltransferase(MtaA), 4) Methyltetrahydrofolate:corrinoid protein methyltransferase(AcsE), 5) Corrinoid iron-sulfur protein (AcsD), 6) Carbon monoxidedehydrogenase (AcsA), 7) Nickel-protein assembly protein (AcsF & CooC),8) Ferredoxin (Orf7), 9) Acetyl-CoA synthase (AcsB & AcsC), 10) Pyruvateferredoxin oxidoreductase (Por), 11) Pyruvate dehydrogenase (PDH), 12)Pyruvate formate lyase (Pfl), 13) Formate dehydrogenase, 14)Acetoacetyl-CoA thiolase (AtoB), 15) Acetoacetyl-CoA:acetate:CoAtransferase (AtoAD), 16) Acetoacetate decarboxylase (Adc), and 17)Isopropanol dehydrogenase (Adh); when glucose and methanol are fed in1.2 ratio, it provides an increase from 1 mol isopropanol/mol glucose to2 mol isopropanol/mol glucose.

FIG. 7a shows flux distribution for improvement in 1,4-BDO yields fromcarbohydrates when reducing equivalents from syngas components areavailable; the enzymatic transformations shown are carried out by thefollowing enzymes: 1) Hydrogenase, 2) Carbon monoxide dehydrogenase, 3)Succinyl-CoA transferase, Succinyl-CoA hydrolase, or Succinyl-CoAsynthetase (or succinyl-CoA ligase), 4) Succinyl-CoA reductase (aldehydeforming), 5) 4-Hydroxybutyrate dehydrogenase, 6) 4-Hydroxybutyratekinase, 7) Phosphotrans-4-hydroxybutyrylase, 8) 4-Hydroxybutyryl-CoAreductase (aldehyde forming), 9) 1,4-butanediol dehydrogenase, 10)Succinate reductase, 11) Succinyl-CoA reductase (alcohol forming), 12)4-Hydroxybutyryl-CoA transferase, 4-Hydroxybutyryl-CoA hydrolase, or4-Hydroxybutyryl-CoA synthetase, 13) 4-Hydroxybutyrate reductase, 14)4-Hydroxybutyryl-phosphate reductase, and 15) 4-Hydroxybutyryl-CoAreductase (alcohol forming).

FIG. 7b shows flux distribution for improvement in 1,3-BDO yields fromcarbohydrates when reducing equivalents from syngas components areavailable; the enzymatic transformations shown are carried out by thefollowing enzymes: 1) Hydrogenase, 2) Carbon monoxide dehydrogenase, 3)Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoAligase), 4) Succinyl-CoA reductase (aldehyde forming), 5)4-Hydroxybutyrate dehydrogenase, 6) 4-Hydroxybutyrate kinase, 7)Phosphotrans-4-hydroxybutyrylase, 8) 4-Hydroxybutyryl-CoA dehydratase,9) crotonase, 10) 3-Hydroxybutyryl-CoA reductase (aldehyde forming), 11)3-Hydroxybutyraldehyde reductase, 12) Succinate reductase, 13)Succinyl-CoA reductase (alcohol forming), 14) 4-Hydroxybutyryl-CoAtransferase, or 4-Hydroxybutyryl-CoA synthetase, 15)3-Hydroxybutyryl-CoA reductase (alcohol forming), 16)3-Hydroxybutyryl-CoA hydrolase, or 3-Hydroxybutyryl-CoA synthetase, or3-Hydroxybutyryl-CoA transferase, and 17) 3-Hydroxybutyrate reductase.

FIG. 7c shows flux distribution for improvement in butanol yields oncarbohydrates when reducing equivalents from syngas components areavailable; the enzymatic transformations shown are carried out by thefollowing enzymes: 1) Hydrogenase, 2) Carbon monoxide dehydrogenase, 3)Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoAligase), 4) Succinyl-CoA reductase (aldehyde forming), 5)4-Hydroxybutyrate dehydrogenase, 6) 4-Hydroxybutyrate kinase, 7)Phosphotrans-4-hydroxybutyrylase, 8) 4-Hydroxybutyryl-CoA dehydratase,9) butyryl-CoA dehydrogenase, 10) Butyryl-CoA reductase (aldehydeforming), 11) Butyraldehyde reductase, 12) Succinate reductase, 13)Succinyl-CoA reductase (alcohol forming), 14) 4-Hydroxybutyryl-CoAtransferase, or 4-Hydroxybutyryl-CoA synthetase, 15) Butyryl-CoAreductase (alcohol forming), 16) Butyryl-CoA hydrolase, or Butyryl-CoAsynthetase, or Butyryl-CoA transferase, and 17) Butyrate reductase.

FIG. 7d shows flux distribution for improvement in yields of6-aminocaproic acid and hexamethylene diamine on carbohydrates whenreducing equivalents from syngas components are available; the enzymatictransformations shown are carried out by the following enzymes: 1)Hydrogenase, 2) Carbon monoxide dehydrogenase, 3) 3-Oxoadipyl-CoAthiolase, 4) 3-Oxoadipyl-CoA reductase, 5) 3-Hydroxyadipyl-CoAdehydratase, 6) 5-Carboxy-2-pentenoyl-CoA reductase, 7) Adipyl-CoAreductase (aldehyde forming), 8) 6-Aminocaproate transaminase, or6-Aminocaproate dehydrogenase, 9) 6-Aminocaproyl-CoA/acyl-CoAtransferase, or 6-Aminocaproyl-CoA synthase, 10) Amidohydrolase, 11)Spontaneous cyclization, 12) 6-Aminocaproyl-CoA reductase (aldehydeforming), and 13) HMDA transaminase, or HMDA dehydrogenase.

FIG. 7e shows flux distribution for improvement in yields of glyceroland 1,3-propanediol on carbohydrates when reducing equivalents fromsyngas components are available; the enzymatic transformations shown arecarried out by the following enzymes: 1) Hydrogenase, 2) Carbon monoxidedehydrogenase, 3) Dihydroxyacetone kinase, 4) Glycerol dehydrogenase, 5)Glycerol dehydratase, 6) 1,3-Propanediol dehydrogenase.

FIG. 8 shows the pathway for the reverse TCA cycle coupled with carbonmonoxide dehydrogenase and hydrogenase for the conversion of syngas toacetyl-CoA.

FIG. 9 shows Western blots of 10 micrograms ACS90 (lane 1), ACS91(lane2), Mta98/99 (lanes 3 and 4) cell extracts with size standards(lane 5) and controls of M. thermoacetica CODH (Moth_1202/1203) or Mtr(Moth_1197) proteins (50, 150, 250, 350, 450, 500, 750, 900, and 1000ng).

FIG. 10 shows CO oxidation assay results. Cells (M. thermoacetica or E.coli with the CODH/ACS operon; ACS90 or ACS91 or empty vector: pZA33S)were grown and extracts prepared. Assays were performed at 55° C. atvarious times on the day the extracts were prepared. Reduction ofmethylviologen was followed at 578 nm over a 120 sec time course.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed, in part to engineered biosynthetic pathwaysto improve carbon flux through the central metabolism intermediate,acetyl-CoA, en route to product molecules. Exemplary product moleculesinclude, without limitation, 1,3-butanediol, isopropanol,4-hydroxybutyrate, and 1,4-butanediol, although given the teachings andguidance provided herein, it will be recognized by one skilled in theart that any product molecule that has acetyl-CoA as a building blockcan exhibit enhanced production through increased carbon flux throughacetyl-CoA. The present invention provides non-naturally occurringmicrobial organisms having one or more exogenous genes encoding enzymesthat can catalyze various enzymatic transformations en route toacetyl-CoA. In some embodiments, these enzymatic transformations arepart of the reductive tricarboxylic acid (RTCA) cycle and are used toimprove product yields from carbohydrate-based carbon feedstock.

This invention is also directed, in part, to improving product yieldsbased on enzymatic transformations of the Wood-Ljungdahl pathway. Insome embodiments, syngas components, such as CO and H₂, can serve assource of reducing equivalents. Such reducing equivalents can improveproduct yields from carbohydrate-based carbon feedstock as describedherein below.

In numerous engineered pathways, realization of maximum product yieldsbased on carbohydrate feedstock is hampered by insufficient reducingequivalents or by loss of reducing equivalents and/or carbon tobyproducts. In accordance with some embodiments, the present inventionincreases the yields of products by (i) enhancing carbon fixation viathe Wood-Ljungdahl pathway and/or the reductive TCA cycle, and (ii)accessing additional reducing equivalents from gaseous syngas componentssuch as CO, CO₂, and/or H₂. Products that can be produced bynon-naturally occurring organisms and methods described herein include,without limitation, ethanol, butanol, isobutanol, 1,3-butanediol,isopropanol, 4-hydroxybutyrate, 1,4-butanediol, succinic acid, fumaricacid, malic acid, 3-hydroxypropionic acid, lactic acid, adipic acid,6-aminocaproic acid, hexamethylenediamine, caprolactam,3-hydoxyisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid,acrylic acid, 1,3-propanediol, glycerol, and long chain hydrocarbons,alcohols, acids, and esters.

The CO₂-fixing reductive tricarboxylic acid (RTCA) cycle is anendergenic anabolic pathway of CO₂ assimilation which uses NAD(P)H andATP (FIG. 2a ). One turn of the RTCA cycle assimilates two moles of CO₂into one mole of acetyl-CoA, or four moles of CO₂ into one mole ofoxaloacetate. This additional availability of acetyl-CoA improves themaximum theoretical yield of product molecules derived fromcarbohydrate-based carbon feedstock. Exemplary carbohydrates include butare not limited to glucose, sucrose, xylose, arabinose and glycerol.Note that the pathways for the exemplary product molecules describedherein all proceed through acetyl-CoA. For example, the fixation of CO₂provides 2.67 molecules of acetyl-CoA from every molecule of glucose,thus improving the maximum product yields as follows:

C₆H₁₂O₆→1.091C₄H₁₀O₂+1.636CO₂+0.545H₂O  1,3-Butanediol:

C₆H₁₂O₆→1.333C₃H₈O+2CO₂+0.667H₂O  Isopropanol:

C₆H₁₂O₆→1.333C₄H₈O₃+0.667CO₂+0.667H₂O  4-Hydroxybutyate:

C₆H₁₂O₆→1.091C₄H₁₀O₂+1.636CO₂+0.545H₂O  1,4-Butanediol:

FIG. 2b provides an exemplary flux distribution showing how the maximumtheoretical isopropanol yield increases from 1 mole/mole glucose to 1.33moles per mole glucose. The reductive TCA cycle was first reported inthe green sulfur photosynthetic bacterium Chlorobium limicola (Evans etal., Proc. Natl. Acad. Sci. U.S.A. 55:928-934 (1966)). Similar pathwayshave been characterized in some prokaryotes (proteobacteria, greensulfur bacteria and thermophillic Knallgas bacteria) andsulfur-dependent archaea (Hugler et al. J. Bacteriol. 187:3020-3027(2005); Hugler et al. Environ. Microbiol. 9:81-92 (2007)). In somecases, reductive and oxidative (Krebs) TCA cycles are present in thesame organism (Hugler et al. supra (2007); Siebers et al. J. Bacteriol.186:2179-2194 (2004). Some methanogens and obligate anaerobes possessincomplete oxidative or reductive TCA cycles that can function tosynthesize biosynthetic intermediates (Ekiel et al. J. Bacteriol.162:905-908 (1985); Wood et al. FEMS Microbiol. 28: 335-352 (2004).

Many of the enzymes in the TCA cycle are reversible and can catalyzereactions in the reductive and oxidative directions. Several reactionsare irreversible and utilize different enzymes to catalyze the forwardand reverse directions. These reactions include: 1) conversion ofcitrate to oxaloacetate and acetyl-CoA, 2) conversion of fumarate tosuccinate, 3) conversion of succinyl-CoA to 2-oxoglutarate. In thecatabolic TCA cycle, citrate is formed from the condensation ofoxaloacetate and acetyl-CoA. The reverse reaction, cleavage of citrateto oxaloacetate and acetyl-CoA, is ATP-dependent and catalyzed by ATPcitrate lyase or citryl-CoA synthetase and citryl-CoA lyase. Theconversion of succinate to fumarate is catalyzed by succinatedehydrogenase while the reverse reaction is catalyzed by fumaratereductase. In the catabolic TCA cycle, succinyl-CoA is formed from theNAD(P)⁺ dependent decarboxylation of 2-oxoglutarate by the AKGDHcomplex. The reverse reaction is catalyzed byalpha-ketoglutarate:ferredoxin oxidoreductase. In some embodiments, theinvention provides non-naturally occurring organisms that enhance carbonflux through acetyl-CoA by engineering one or more enzymes that are partof the reverse TCA cycle.

In some embodiments, the invention provides enhanced product yields viacarbohydrate-based carbon feedstock by fixing carbon dioxide and/ormethanol via the Wood-Ljungdahl pathway or components thereof. Synthesisgas (syngas) is a mixture of H₂ and CO that can be obtained viagasification of any organic feedstock, such as coal, coal oil, naturalgas, biomass, or waste organic matter. Numerous gasification processeshave been developed, and most designs are based on partial oxidation,where limiting oxygen avoids full combustion, of organic materials athigh temperatures (500-1500° C.) to provide syngas as a 0.5:1-3:1 H₂/COmixture. In addition to coal, biomass of many types has been used forsyngas production and this source represents an inexpensive and flexiblefeedstock for the biological production of renewable chemicals andfuels.

There are known pathways in organisms such as Clostridia that utilizesyngas effectively. Specifically, acetogens, such as Moorellathermoacetica, C. ljungdahlii and C. carboxidivorans, can grow on anumber of carbon sources ranging from hexose sugars to carbon monoxide.Hexoses, such as glucose, are metabolized first viaEmbden-Meyerhof-Parnas (EMP) glycolysis to pyruvate, which is thenconverted to acetyl-CoA via pyruvate:ferredoxin oxidoreductase (PFOR).Acetyl-CoA can be used to build biomass precursors or can be convertedto acetate which produces energy via acetate kinase andphosphotransacetylase. The overall conversion of glucose to acetate,energy, and reducing equivalents is

C₆H₁₂O₆+4ADP+4Pi→CH₃COOH+2CO₂+4ATP+8[H]

Acetogens extract even more energy out of the glucose to acetateconversion while also maintaining redox balance by further convertingthe released CO₂ to acetate via the Wood-Ljungdahl pathway:

2CO₂+8[H]+nADP+nPi→2CH₃COOH+nATP

The coefficient “n” in the above equation signifies that this conversionis an energy generating endeavor, as many acetogens can grow in thepresence of CO₂ via the Wood-Ljungdahl pathway even in the absence ofglucose as long as hydrogen is present to supply reducing equivalents.

2CO₂+4H₂ +nADP+nPi→CH₃COOH+2H₂O+nATP

The Wood-Ljungdahl pathway, illustrated in FIG. 3A, is coupled to thecreation of Na⁺ or H⁺ ion gradients that can generate ATP via an Na⁺ orH⁺-dependant ATP synthase, respectively (Muller, V., Appl. Environ.Microbiol. 69:6345-6353 (2003)). Based on these known transformations,acetogens also have the capacity to utilize CO as the sole carbon andenergy source. Specifically, CO can be oxidized to produce reducingequivalents and CO₂, or directly assimilated into acetyl-CoA which issubsequently converted to either biomass or acetate.

4CO+2H₂O CH₃COOH+2CO₂

Even higher acetate yields, however, can be attained when enoughhydrogen is present to satisfy the requirement for reducing equivalents.

2CO+2H₂CH₃COOH

Following from FIG. 3A, the production of acetate via acetyl-CoAgenerates one ATP molecule.

Methanol is a relatively inexpensive organic feedstock that can bederived from synthesis gas components, CO and H₂, via catalysis. Anon-naturally occurring microbial organism of the invention capable ofutilizing methanol can also utilize gases including, for example, CO,CO₂, and/or H₂ for conversion to acetyl-CoA, cell mass, and products.Specifically, acetogens such as Moorella thermoacetica (formerly,Clostridium thermoaceticum) use syngas via the Wood-Ljungdahl pathway.This pathway includes two branches: the Eastern (or methyl) branchconverts CO₂ to methyltetrahydrofolate (Me-THF) and the Western (orcarbonyl) branch that converts methyl-THF, CO, and Coenzyme-A intoacetyl-CoA (FIG. 3B). Any non-naturally occurring microorganism of theinvention expressing genes encoding enzymes that catalyze thecarbonyl-branch of the Wood-Ljungdahl pathway in conjunction with aMtaABC-type methyltransferase system is capable of ‘fixing’ carbon fromexogenous CO and/or CO₂ and methanol to synthesize acetyl-CoA, cellmass, and products.

Implementing the pathway to form acetyl-CoA from methanol and syngas,heretofor referred to as the “methanol Wood-Ljungdahl pathway,” isenergetically favorable compared to utilizing the full Wood-Ljungdahlpathway. For example, the direct conversion of synthesis gas to acetateis an energetically neutral process (see FIG. 3B). Specifically, one ATPmolecule is consumed during the formation of formyl-THF by formyl-THFsynthase and one ATP molecule is produced during the production ofacetate via acetate kinase. ATP consumption can be circumvented byensuring that the methyl group on the methyl branch product, methyl-THF,is obtained from methanol rather than CO₂. The result is that acetateformation has a positive ATP yield that can help support cell growth andmaintenance. A non-naturally occurring microbial organism of the presentinvention, engineered with these capabilities, that also naturallypossesses the capability for anapleurosis (e.g., E. coli) can grow onthe methanol and syngas-generated acetyl-CoA in the presence of asuitable external electron acceptor such as nitrate. This electronacceptor is used to accept electrons from the reduced quinone formed viasuccinate dehydrogenase. A further use of adding an external electronacceptor is that additional energy for cell growth, maintenance, andproduct formation can be generated from respiration of acetyl-CoA. Insome embodiments, engineering a pyruvate ferredoxin oxidoreductase(PFOR) enzyme into a non-naturally occurring microbial organism allowsthe synthesis of biomass precursors in the absence of an externalelectron acceptor.

Carbon from syngas and/or methanol can be fixed via the Wood-Ljungdahlpathway and portions thereof when using carbohydrate-based carbonfeedstock for the formation of molecules such as 1,3-butanediol,isopropanol, 4-hydroxybutyrate, and 1,4-butanediol using the pathwaysdescribed herein. Specifically, the combination of certainsyngas-utilization pathway components with the acetyl-CoA to1,3-butanediol, isopropanol, 4-hydroxybutyrate, or 1,4-butanediolpathways results in high yields of these products from carbohydrates byproviding an efficient mechanism for fixing the carbon present in carbondioxide, fed exogenously or produced endogenously, into acetyl-CoA asshown below. Exemplary carbohydrates include but are not limited toglucose, sucrose, xylose, arabinose and glycerol. The enzymatictransformations for carbon fixation are shown in FIGS. 4A and 4Brespectively.

C₆H₁₂O₆→1.091C₄H₁₀O₂+1.636CO₂+0.545H₂O  1,3-Butanediol:

C₆H₁₂O₆→1.333C₃H₈O+2CO₂+0.667H₂O  Isopropanol:

C₆H₁₂O₆→1.333C₄H₈O₃+0.667CO₂+0.667H₂O  4-Hydroxybutyate:

C₆H₁₂O₆→1.091C₄H₁₀O₂+1.636CO₂+0.545H₂O  1,4-Butanediol:

The maximum theoretical yields of isopropanol, 4-hydroxybutyrate, and1,4-butanediol from synthesis gases or carbohydrates can be furtherenhanced by the addition of methanol in different ratios of methanol toglucose. This is shown in the equations below:

CH₄O+C₆H₁₂O₆→1.364C₄H₁₀O₂+1.545CO₂+1.182H₂O  1,3-Butanediol:

CH₄O+C₆H₁₂O₆→1.667C₃H₈O+2CO₂+1.333H₂O  Isopropanol:

CH₄O+C₆H₁₂O₆→1.667C₄H₈O₃+0.333CO₂+1.333H₂O  4-Hydroxybutyate:

CH₄O+C₆H₁₂O₆→1.364C₄H₁₀O₂+1.545CO₂+1.182H₂O  1,4-Butanediol:

2CH₄O+C₆H₁₂O₆→1.636C₄H₁₀O₂+1.455CO₂+1.818H₂O  1,3-Butanediol:

2CH₄O+C₆H₁₂O₆→2C₃H₈O+2CO₂+2H₂O  Isopropanol:

2CH₄O+C₆H₁₂O₆→2C₄H₈O₃+2H₂O  4-Hydroxybutyate:

2CH₄O+C₆H₁₂O₆→1.636C₄H₁₀O₂+1.455CO₂+1.818H₂O  1,4-Butanediol:

Exemplary flux distributions showing improvements in yields of1,3-butanediol and isopropanol via carbohydrate-based carbon feedstockwhen carbon can be fixed via the Wood-Ljungdahl pathway using syngascomponents with and without methanol are shown in FIGS. 5 and 6,respectively.

Thus, the non-naturally occurring microbial organisms and conversionroutes described herein provide an efficient means of convertingcarbohydrates to products such as isopropanol, 4-hydroxybutyrate, or1,4-butanediol. Additional product molecules that can be produced by theteachings of this invention include but are not limited to ethanol,butanol, isobutanol, isopropanol, 1,4-butanediol, succinic acid, fumaricacid, malic acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, lacticacid, adipic acid, 6-aminocaproic acid, hexamethylenediamine,caprolactam, 3-hydoxyisobutyric acid, 2-hydroxyisobutyric acid,methacrylic acid, acrylic acid, glycerol, 1,3-propanediol, and longchain hydrocarbons, alcohols, acids, and esters.

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial organism's genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within a1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, isopropanol,6-aminocaproic acid, hexamethylene diamine, caprolactam, glycerol, or1,3-propanediol biosynthetic pathway.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides, or functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” are intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

It is understood that when more than one exogenous nucleic acid isincluded in a microbial organism that the more than one exogenousnucleic acids refers to the referenced encoding nucleic acid orbiosynthetic activity, as discussed above. It is further understood, asdisclosed herein, that such more than one exogenous nucleic acids can beintroduced into the host microbial organism on separate nucleic acidmolecules, on polycistronic nucleic acid molecules, or a combinationthereof, and still be considered as more than one exogenous nucleicacid. For example, as disclosed herein a microbial organism can beengineered to express two or more exogenous nucleic acids encoding adesired pathway enzyme or protein. In the case where two exogenousnucleic acids encoding a desired activity are introduced into a hostmicrobial organism, it is understood that the two exogenous nucleicacids can be introduced as a single nucleic acid, for example, on asingle plasmid, on separate plasmids, can be integrated into the hostchromosome at a single site or multiple sites, and still be consideredas two exogenous nucleic acids. Similarly, it is understood that morethan two exogenous nucleic acids can be introduced into a host organismin any desired combination, for example, on a single plasmid, onseparate plasmids, can be integrated into the host chromosome at asingle site or multiple sites, and still be considered as two or moreexogenous nucleic acids, for example three exogenous nucleic acids.Thus, the number of referenced exogenous nucleic acids or biosyntheticactivities refers to the number of encoding nucleic acids or the numberof biosynthetic activities, not the number of separate nucleic acidsintroduced into the host organism.

The non-naturally occurring microbial organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, isopropanol, 6-aminocaproic acid,hexamethylene diamine, caprolactam, glycerol, or 1,3-propanediolbiosynthetic capability, those skilled in the art will understand withapplying the teaching and guidance provided herein to a particularspecies that the identification of metabolic modifications can includeidentification and inclusion or inactivation of orthologs. To the extentthat paralogs and/or nonorthologous gene displacements are present inthe referenced microorganism that encode an enzyme catalyzing a similaror substantially similar metabolic reaction, those skilled in the artalso can utilize these evolutionary related genes.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

In some embodiments, the present invention provides a non-naturallyoccurring microbial organism that includes a microbial organism having areductive TCA pathway in which at least one exogenous nucleic acidencoding a reductive TCA pathway enzyme is expressed in a sufficientamount to enhance carbon flux through acetyl-CoA. At least one exogenousnucleic acid is selected from an ATP-citrate lyase, citrate lyase, afumarate reductase, and an alpha-ketoglutarate:ferredoxinoxidoreductase.

In some embodiments, the non-naturally occurring microbial organismincludes two exogenous nucleic acids each encoding a reductive TCApathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes three exogenous nucleic acids each encoding a reductive TCApathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes three exogenous nucleic acids encoding an ATP-citrate lyase, afumarate reductase, and an alpha-ketoglutarate:ferredoxinoxidoreductase.

In some embodiments, the non-naturally occurring microbial organismincludes three exogenous nucleic acids encoding a citrate lyase, afumarate reductase, and an alpha-ketoglutarate:ferredoxinoxidoreductase.

In some embodiments, the non-naturally occurring microbial organismincludes at least one exogenous nucleic acid that is a heterologousnucleic acid.

In some embodiments, the non-naturally occurring microbial organism isin a substantially anaerobic culture medium.

In some embodiments, the non-naturally occurring microbial organismfurther includes an exogenous nucleic acid encoding an enzyme selectedfrom a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitratedehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, afumarase, a malate dehydrogenase, an acetate kinase, aphosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxinoxidoreductase, and combinations thereof.

In some embodiments, the non-naturally occurring microbial organismfurther induces an isopropanol pathway, the isopropanol pathwayconverting acetyl-CoA to isopropanol, wherein the isopropanol pathwayincludes 1) an acetoacetyl-CoA thiolase, 2) an acetoacetyl-CoAtransferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoAsynthetase, or a phosphotransacetoacetylase/acetoacetate kinase, 3) anacetoacetate decarboxylase, and 4) an isopropanol dehydrogenase.

In some embodiments, the non-naturally occurring microbial organismincludes at least one enzyme of the isopropanol pathway which is encodedby an exogenous nucleic acid.

In some embodiments, the non-naturally occurring microbial organismincludes at least two enzymes of the isopropanol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least three enzymes of the isopropanol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes four enzymes of the isopropanol pathway which are encoded byexogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismfurther includes a 1,3-butanediol pathway; said 1,3-butanediol pathwayconverting acetyl-CoA to 1,3-butanediol, wherein said 1,3-butanediolpathway comprises at least three enzymes selected from 1)Acetoacetyl-CoA thiolase (AtoB), 2) Acetoacetyl-CoA reductase(CoA-dependent, alcohol forming), 3) 3-oxobutyraldehyde reductase(aldehyde reducing), 4) 4-hydroxy,2-butanone reductase, 5)Acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), 6)3-oxobutyraldehyde reductase (ketone reducing), 7)3-hydroxybutyraldehyde reductase, 8) Acetoacetyl-CoA reductase (ketonereducing), 9) 3-hydroxybutyryl-CoA reductase (aldehyde forming), 10)3-hydroxybutyryl-CoA reductase (alcohol forming), 11) an acetoacetyl-CoAtransferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoAsynthetase, or a phosphotransacetoacetylase/acetoacetate kinase, 12)Acetoacetate reductase, 13) 3-hydroxybutyryl-CoA transferase, hydrolase,or synthetase, 14) 3-hydroxybutyrate reductase, and 15)3-hydroxybutyrate dehydrogenase.

In some embodiments, the non-naturally occurring microbial organismincludes at least one enzyme of the 1,3-butanediol pathway which isencoded by an exogenous nucleic acid.

In some embodiments, the non-naturally occurring microbial organismincludes at least two enzymes of the 1,3-butanediol pathway are encodedby exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least three enzymes of the 1,3-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismfurther includes a 1,4-butanediol pathway, the 1,4-butanediol pathwayconverting acetyl-CoA to 1,4-butanediol, wherein the 1,4-butanediolpathway includes at least five enzymes selected from 1) Acetoacetyl-CoAthiolase (AtoB), 2) 3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3)Crotonase (Crt), 4) Crotonyl-CoA hydratase (4-Budh), 5)4-hydroxybutyryl-CoA reductase (alcohol forming), 6)4-hydroxybutyryl-CoA reductase (aldehyde forming), 7) 1,4-butanedioldehydrogenase, 8) 4-Hydroxybutyryl-CoA transferase, 4-Hydroxybutyryl-CoAsynthetase, 4-Hydroxybutyryl-CoA hydrolase, orPhosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate kinase, and 9)4-Hydroxybutyrate reductase.

In some embodiments, the non-naturally occurring microbial organismincludes at least one enzyme of the 1,4-butanediol pathway which isencoded by an exogenous nucleic acid.

In some embodiments, the non-naturally occurring microbial organismincludes at least two enzymes of the 1,4-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least three enzymes of the 1,4-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least four enzymes of the 1,4-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least five enzymes of said 1,4-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismfurther includes a 4-hydroxybutyrate pathway, the 4-hydroxybutyratepathway converting acetyl-CoA to 4-hydroxybutyrate, wherein the4-hydroxybutyrate pathway includes at least five enzymes selectedfrom 1) Acetoacetyl-CoA thiolase (AtoB), 2) 3-Hydroxybutyryl-CoAdehydrogenase (Hbd), 3) Crotonase (Crt), 4) Crotonyl-CoA hydratase(4-Budh), 5) 4-Hydroxybutyryl-CoA transferase, hydrolase or synthetase,6) Phosphotrans-4-hydroxybutyrylase, and 7) 4-Hydroxybutyrate kinase.

In some embodiments, the non-naturally occurring microbial organismincludes at least one enzyme of the 4-hydroxybutyrate pathway which isencoded by an exogenous nucleic acid.

In some embodiments, the non-naturally occurring microbial organismincludes at least two enzymes of the 4-hydroxybutyrate pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least three enzymes of the 4-hydroxybutyrate pathway whichare encoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least four enzymes of the 4-hydroxybutyrate pathway whichare encoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least five enzymes of the 4-hydroxybutyrate pathway whichare encoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismfurther includes an exogenous nucleic acid encoding an enzyme selectedfrom carbon monoxide dehydrogenase, acetyl-CoA synthase, ferredoxin,NAD(P)H:ferredoxin oxidoreductase and combinations thereof.

In some embodiments, the non-naturally occurring microbial organismutilizes a carbon feedstock selected from CO, CO₂, CO₂ and H₂, synthesisgas comprising CO and H₂, and synthesis gas comprising CO, CO₂, and H₂.

In some embodiments, a non-naturally occurring microbial organismincludes a microbial organism having a Wood-Ljungdahl pathway thatincludes at least one exogenous nucleic acid encoding a Wood-Ljungdahlpathway enzyme expressed in a sufficient amount to enhance carbon fluxthrough acetyl-CoA. The at least one exogenous nucleic acid is selectedfrom a) Formate dehydrogenase, b) Formyltetrahydrofolate synthetase, c)Methenyltetrahydrofolate cyclohydrolase, d) Methylenetetrahydrofolatedehydrogenase, e) Methylenetetrahydrofolate reductase,Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), g)Corrinoid iron-sulfur protein (AcsD), h) Nickel-protein assembly protein(AcsF & CooC), i) Ferredoxin (Orf7), j) Acetyl-CoA synthase (AcsB &AcsC), k) Carbon monoxide dehydrogenase (AcsA), 1) Pyruvate ferredoxinoxidoreductase or pyruvate dehydrogenase, m) Pyruvate formate lyase

In some embodiments, the non-naturally occurring microbial organismincludes two exogenous nucleic acids each encoding a Wood-Ljungdahlpathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes three exogenous nucleic acids each encoding a Wood-Ljungdahlpathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes four exogenous nucleic acids each encoding a Wood-Ljungdahlpathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes five exogenous nucleic acids each encoding a Wood-Ljungdahlpathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes six exogenous nucleic acids each encoding a Wood-Ljungdahlpathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes seven exogenous nucleic acids each encoding a Wood-Ljungdahlpathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes eight exogenous nucleic acids each encoding a Wood-Ljungdahlpathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes nine exogenous nucleic acids each encoding a Wood-Ljungdahlpathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes ten exogenous nucleic acids each encoding a Wood-Ljungdahlpathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes eleven exogenous nucleic acids each encoding a Wood-Ljungdahlpathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes twelve exogenous nucleic acids each encoding a Wood-Ljungdahlpathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes at least one exogenous nucleic acid that is a heterologousnucleic acid.

In some embodiments, the non-naturally occurring microbial organismincludes is in a substantially anaerobic culture medium.

In some embodiments, the non-naturally occurring microbial organismfurther includes an isopropanol pathway, the isopropanol pathwayconverting acetyl-CoA to isopropanol. The isopropanol pathwayincludes 1) an acetoacetyl-CoA thiolase, 2) an acetoacetyl-CoAtransferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoAsynthetase, or a phosphotransacetoacetylase/acetoacetate kinase, 3) anacetoacetate decarboxylase, and 4) an isopropanol dehydrogenase.

In some embodiments, the non-naturally occurring microbial organismincludes at least one enzyme of the isopropanol pathway that is encodedby an exogenous nucleic acid.

In some embodiments, the non-naturally occurring microbial organismincludes at least two enzymes of the isopropanol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least three enzymes of the isopropanol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes the four enzymes of the isopropanol pathway which are encodedby exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismfurther includes a 1,3-butanediol pathway; the 1,3-butanediol pathwayconverting acetyl-CoA to 1,3-butanediol. The 1,3-butanediol pathwayincludes at least three enzymes selected from 1) Acetoacetyl-CoAthiolase (AtoB), 2) Acetoacetyl-CoA reductase (CoA-dependent, alcoholforming), 3) 3-oxobutyraldehyde reductase (aldehyde reducing), 4)4-hydroxy,2-butanone reductase, 5) Acetoacetyl-CoA reductase(CoA-dependent, aldehyde forming), 6) 3-oxobutyraldehyde reductase(ketone reducing), 7) 3-hydroxybutyraldehyde reductase, 8)Acetoacetyl-CoA reductase (ketone reducing), 9) 3-hydroxybutyryl-CoAreductase (aldehyde forming), 10) 3-hydroxybutyryl-CoA reductase(alcohol forming), 11) an acetoacetyl-CoA transferase, anacetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or aphosphotransacetoacetylase/acetoacetate kinase, 12) Acetoacetatereductase, 13) 3-hydroxybutyryl-CoA transferase, hydrolase, orsynthetase, 14) 3-hydroxybutyrate reductase, and 15) 3-hydroxybutyratedehydrogenase.

In some embodiments, the non-naturally occurring microbial organismincludes at least one enzyme of the 1,3-butanediol pathway that isencoded by an exogenous nucleic acid.

In some embodiments, the non-naturally occurring microbial organismincludes at least two enzymes of the 1,3-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least three enzymes of the 1,3-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismfurther includes a 1,4-butanediol pathway, the 1,4-butanediol pathwayconverting acetyl-CoA to 1,4-butanediol. The 1,4-butanediol pathwayincludes at least five enzymes selected from 1) Acetoacetyl-CoA thiolase(AtoB), 2) 3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3) Crotonase (Crt),4) Crotonyl-CoA hydratase (4-Budh), 5) 4-hydroxybutyryl-CoA reductase(alcohol forming), 6) 4-hydroxybutyryl-CoA reductase (aldehyde forming),7) 1,4-butanediol dehydrogenase, 8) 4-Hydroxybutyryl-CoA transferase,4-Hydroxybutyryl-CoA synthetase, 4-Hydroxybutyryl-CoA hydrolase, orPhosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate kinase, and 9)4-Hydroxybutyrate reductase.

In some embodiments, the non-naturally occurring microbial organismincludes at least one enzyme of the 1,4-butanediol pathway that isencoded by an exogenous nucleic acid.

In some embodiments, the non-naturally occurring microbial organismincludes at least two enzymes of the 1,4-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least three enzymes of the 1,4-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least four enzymes of the 1,4-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least five enzymes of the 1,4-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismfurther includes a 4-hydroxybutyrate pathway, the 4-hydroxybutyratepathway converting acetyl-CoA to 4-hydroxybutyrate. The4-hydroxybutyrate pathway includes at least five enzymes selectedfrom 1) Acetoacetyl-CoA thiolase (AtoB), 2) 3-Hydroxybutyryl-CoAdehydrogenase (Hbd), 3) Crotonase (Crt), 4) Crotonyl-CoA hydratase(4-Budh), 5) 4-Hydroxybutyryl-CoA transferase, hydrolase or synthetase,6) Phosphotrans-4-hydroxybutyrylase, and 7) 4-Hydroxybutyrate kinase.

In some embodiments, the non-naturally occurring microbial organismincludes at least one enzyme of the 4-hydroxybutyrate pathway that isencoded by an exogenous nucleic acid.

In some embodiments, the non-naturally occurring microbial organismincludes at least two enzymes of the 4-hydroxybutyrate pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least three enzymes of the 4-hydroxybutyrate pathway whichare encoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least four enzymes of the 4-hydroxybutyrate pathway whichare encoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least five enzymes of the 4-hydroxybutyrate pathway whichare encoded by exogenous nucleic acids.

In some embodiments, a non-naturally occurring microbial organismincludes a microbial organism having a methanol Wood-Ljungdahl pathwaythat includes at least one exogenous nucleic acid encoding aWood-Ljungdahl pathway enzyme expressed in a sufficient amount toenhance carbon flux through acetyl-CoA. The at least one exogenousnucleic acid is selected from a) Methanol methyltransferase (MtaB), b)Corrinoid protein (MtaC), c) Methyltetrahydrofolate:corrinoid proteinmethyltransferase (MtaA), d) Methyltetrahydrofolate:corrinoid proteinmethyltransferase (AcsE), e) Corrinoid iron-sulfur protein (AcsD), f)Nickel-protein assembly protein (AcsF & CooC), g) Ferredoxin (Orf7), h)Acetyl-CoA synthase (AcsB & AcsC), i) Carbon monoxide dehydrogenase(AcsA), j) Pyruvate ferredoxin oxidoreductase, k) NAD(P)H:ferredoxinoxidoreductase, 1) Pyruvate dehydrogenase, m) Pyruvate formate lyase, n)Formate dehydrogenase.

In some embodiments, the non-naturally occurring microbial organismincludes two exogenous nucleic acids each encoding a methanolWood-Ljungdahl pathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes three exogenous nucleic acids each encoding a methanolWood-Ljungdahl pathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes four exogenous nucleic acids each encoding a methanolWood-Ljungdahl pathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes five exogenous nucleic acids each encoding a methanolWood-Ljungdahl pathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes six exogenous nucleic acids each encoding a methanolWood-Ljungdahl pathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes seven exogenous nucleic acids each encoding a methanolWood-Ljungdahl pathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes eight exogenous nucleic acids each encoding a methanolWood-Ljungdahl pathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes nine exogenous nucleic acids each encoding a methanolWood-Ljungdahl pathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes ten exogenous nucleic acids each encoding a methanolWood-Ljungdahl pathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes eleven exogenous nucleic acids each encoding a methanolWood-Ljungdahl pathway enzyme.

In some embodiments, the non-naturally occurring microbial organismincludes at least one exogenous nucleic acid that is a heterologousnucleic acid.

In some embodiments, the non-naturally occurring microbial organism isin a substantially anaerobic culture medium.

In some embodiments, the non-naturally occurring microbial organismfurther includes an isopropanol pathway, the isopropanol pathwayconverting acetyl-CoA to isopropanol. The isopropanol pathwayincludes 1) an acetoacetyl-CoA thiolase, 2) an acetoacetyl-CoAtransferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoAsynthetase, or a phosphotransacetoacetylase/acetoacetate kinase, 3) anacetoacetate decarboxylase, and 4) an isopropanol dehydrogenase.

In some embodiments, the non-naturally occurring microbial organismincludes at least one enzyme of the isopropanol pathway that is encodedby an exogenous nucleic acid.

In some embodiments, the non-naturally occurring microbial organismincludes at least two enzymes of the isopropanol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least three enzymes of the isopropanol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes four enzymes of the isopropanol pathway which are encoded byexogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismfurther includes a 1,3-butanediol pathway, the 1,3-butanediol pathwayconverting acetyl-CoA to 1,3-butanediol. The 1,3-butanediol pathwayincludes at least three enzymes selected from 1) Acetoacetyl-CoAthiolase (AtoB), 2) Acetoacetyl-CoA reductase (CoA-dependent, alcoholforming), 3) 3-oxobutyraldehyde reductase (aldehyde reducing), 4)4-hydroxy,2-butanone reductase, 5) Acetoacetyl-CoA reductase(CoA-dependent, aldehyde forming), 6) 3-oxobutyraldehyde reductase(ketone reducing), 7) 3-hydroxybutyraldehyde reductase, 8)Acetoacetyl-CoA reductase (ketone reducing), 9) 3-hydroxybutyryl-CoAreductase (aldehyde forming), 10) 3-hydroxybutyryl-CoA reductase(alcohol forming), 11) an acetoacetyl-CoA transferase, anacetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or aphosphotransacetoacetylase/acetoacetate kinase, 12) Acetoacetatereductase, 13) 3-hydroxybutyryl-CoA transferase, hydrolase, orsynthetase, 14) 3-hydroxybutyrate reductase, and 15) 3-hydroxybutyratedehydrogenase.

In some embodiments, the non-naturally occurring microbial organismincludes at least one enzyme of the 1,3-butanediol pathway that isencoded by an exogenous nucleic acid.

In some embodiments, the non-naturally occurring microbial organismincludes at least two enzymes of the 1,3-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least three enzymes of the 1,3-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismfurther includes a 1,4-butanediol pathway, the 1,4-butanediol pathwayconverting acetyl-CoA to 1,4-butanediol. The 1,4-butanediol pathwayincludes at least five enzymes selected from 1) Acetoacetyl-CoA thiolase(AtoB), 2) 3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3) Crotonase (Crt),4) Crotonyl-CoA hydratase (4-Budh), 5) 4-hydroxybutyryl-CoA reductase(alcohol forming), 6) 4-hydroxybutyryl-CoA reductase (aldehyde forming),7) 1,4-butanediol dehydrogenase, 8) 4-Hydroxybutyryl-CoA transferase,4-Hydroxybutyryl-CoA synthetase, 4-Hydroxybutyryl-CoA hydrolase, orPhosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate kinase, and 9)4-Hydroxybutyrate reductase.

In some embodiments, the non-naturally occurring microbial organismincludes at least one enzyme of the 1,4-butanediol pathway that isencoded by an exogenous nucleic acid.

In some embodiments, the non-naturally occurring microbial organismincludes at least two enzymes of the 1,4-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least three enzymes of the 1,4-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least four enzymes of the 1,4-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least five enzymes of the 1,4-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismfurther includes a 4-hydroxybutyrate pathway, the 4-hydroxybutyratepathway converting acetyl-CoA to 4-hydroxybutyrate. The4-hydroxybutyrate pathway includes at least five enzymes selectedfrom 1) Acetoacetyl-CoA thiolase (AtoB), 2) 3-Hydroxybutyryl-CoAdehydrogenase (Hbd), 3) Crotonase (Crt), 4) Crotonyl-CoA hydratase(4-Budh), 5) 4-Hydroxybutyryl-CoA transferase, hydrolase or synthetase,6) Phosphotrans-4-hydroxybutyrylase, and 7) 4-Hydroxybutyrate kinase.

In some embodiments, the non-naturally occurring microbial organismincludes at least one enzyme of the 4-hydroxybutyrate pathway that isencoded by an exogenous nucleic acid.

In some embodiments, the non-naturally occurring microbial organismincludes at least two enzymes of the 4-hydroxybutyrate pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least three enzymes of the 4-hydroxybutyrate pathway whichare encoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least four enzymes of the 4-hydroxybutyrate pathway whichare encoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least five enzymes of the 4-hydroxybutyrate pathway whichare encoded by exogenous nucleic acids.

In some embodiments, the present invention provides a non-naturallyoccurring microbial organism that includes at least one exogenousnucleic acid encoding an enzyme expressed in a sufficient amount toenhance the availability of reducing equivalents in the presence ofcarbon monoxide or hydrogen, thereby increasing the yield ofredox-limited products via carbohydrate-based carbon feedstock. The atleast one exogenous nucleic acid is selected from a carbon monoxidedehydrogenase, a hydrogenase, an NAD(P)H:ferredoxin oxidoreductase, anda ferredoxin.

In some embodiments, the non-naturally occurring microbial organismincludes two exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes three exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes four exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes four exogenous nucleic acids encoding a carbon monoxidedehydrogenase, a hydrogenase, an NAD(P)H:ferredoxin oxidoreductase, anda ferredoxin.

In some embodiments, the non-naturally occurring microbial organismincludes at least one exogenous nucleic acid that is a heterologousnucleic acid.

In some embodiments, the non-naturally occurring microbial organism isin a substantially anaerobic culture medium.

In some embodiments, the non-naturally occurring microbial organismfurther includes one or more nucleic acids encoding an enzyme selectedfrom a phosphoenolpyruvate carboxylase, a phosphoenolpyruvatecarboxykinase, a pyruvate carboxylase, and a malic enzyme.

In some embodiments, the non-naturally occurring microbial organismfurther includes one or more nucleic acids encoding an enzyme selectedfrom a malate dehydrogenase, a fumarase, a fumarate reductase, asuccinyl-CoA synthetase, and a succinyl-CoA transferase.

In some embodiments, the non-naturally occurring microbial organismincludes a 1,4-butanediol pathway that includes at least one exogenousnucleic acid encoding an enzyme selected from 1) Hydrogenase, 2) Carbonmonoxide dehydrogenase, 3) Succinyl-CoA transferase, Succinyl-CoAhydrolase, or Succinyl-CoA synthetase (or succinyl-CoA ligase), 4)Succinyl-CoA reductase (aldehyde forming), 5) 4-Hydroxybutyratedehydrogenase, 6) 4-Hydroxybutyrate kinase, 7)Phosphotrans-4-hydroxybutyrylase, 8) 4-Hydroxybutyryl-CoA reductase(aldehyde forming), 9) 1,4-butanediol dehydrogenase, 10) Succinatereductase, 11) Succinyl-CoA reductase (alcohol forming), 12)4-Hydroxybutyryl-CoA transferase, 4-Hydroxybutyryl-CoA hydrolase, or4-Hydroxybutyryl-CoA synthetase, 13) 4-Hydroxybutyrate reductase, 14)4-Hydroxybutyryl-phosphate reductase, and 15) 4-Hydroxybutyryl-CoAreductase (alcohol forming).

In some embodiments, the non-naturally occurring microbial organismfurther includes a 1,3-butanediol pathway comprising at least oneexogenous nucleic acid encoding an enzyme selected from: 1) Hydrogenase,2) Carbon monoxide dehydrogenase, 3) Succinyl-CoA transferase, orSuccinyl-CoA synthetase (or succinyl-CoA ligase), 4) Succinyl-CoAreductase (aldehyde forming), 5) 4-Hydroxybutyrate dehydrogenase, 6)4-Hydroxybutyrate kinase, 7) Phosphotrans-4-hydroxybutyrylase, 8)4-Hydroxybutyryl-CoA dehydratase, 9) Crotonase, 10) 3-Hydroxybutyryl-CoAreductase (aldehyde forming), 11) 3-Hydroxybutyraldehyde reductase, 12)Succinate reductase, 13) Succinyl-CoA reductase (alcohol forming), 14)4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase,15) 3-Hydroxybutyryl-CoA reductase (alcohol forming), 16)3-Hydroxybutyryl-CoA hydrolase, or 3-Hydroxybutyryl-CoA synthetase, or3-Hydroxybutyryl-CoA transferase, 17) 3-Hydroxybutyrate reductase

In some embodiments, the non-naturally occurring microbial organismincludes a butanol pathway that includes at least one exogenous nucleicacid encoding an enzyme selected from 1) Hydrogenase, 2) Carbon monoxidedehydrogenase, 3) Succinyl-CoA transferase, or Succinyl-CoA synthetase(or succinyl-CoA ligase), 4) Succinyl-CoA reductase (aldehyde forming),5) 4-Hydroxybutyrate dehydrogenase, 6) 4-Hydroxybutyrate kinase, 7)Phosphotrans-4-hydroxybutyrylase, 8) 4-Hydroxybutyryl-CoA dehydratase,9) butyryl-CoA dehydrogenase, 10) Butyryl-CoA reductase (aldehydeforming), 11) Butyraldehyde reductase, 12) Succinate reductase, 13)Succinyl-CoA reductase (alcohol forming), 14) 4-Hydroxybutyryl-CoAtransferase, or 4-Hydroxybutyryl-CoA synthetase, 15) Butyryl-CoAreductase (alcohol forming), 16) Butyryl-CoA hydrolase, or Butyryl-CoAsynthetase, or Butyryl-CoA transferase, 17) Butyrate reductase.

In some embodiments, the non-naturally occurring microbial organismfurther includes a 6-aminocaproic acid pathway that includes at leastone exogenous nucleic acid encoding an enzyme selected from 1)Hydrogenase, 2) Carbon monoxide dehydrogenase, 3) 3-Oxoadipyl-CoAthiolase, 4) 3-Oxoadipyl-CoA reductase, 5) 3-Hydroxyadipyl-CoAdehydratase, 6) 5-Carboxy-2-pentenoyl-CoA reductase, 7) Adipyl-CoAreductase (aldehyde forming), and 8) 6-Aminocaproate transaminase, or6-Aminocaproate dehydrogenase.

In some embodiments, the non-naturally occurring microbial organismfurther includes a hexamethylenediamine pathway comprising at least oneexogenous nucleic acid encoding an enzyme selected from 1) Hydrogenase,2) Carbon monoxide dehydrogenase, 3) 3-Oxoadipyl-CoA thiolase, 4)3-Oxoadipyl-CoA reductase, 5) 3-Hydroxyadipyl-CoA dehydratase, 6)5-Carboxy-2-pentenoyl-CoA reductase, 7) Adipyl-CoA reductase (aldehydeforming), 8) 6-Aminocaproate transaminase, or 6-Aminocaproatedehydrogenase, 9) 6-Aminocaproyl-CoA/acyl-CoA transferase, or6-Aminocaproyl-CoA synthase, 10) 6-Aminocaproyl-CoA reductase (aldehydeforming), and 11) HMDA transaminase, or HMDA dehydrogenase.

In some embodiments, the non-naturally occurring microbial organismincludes an adipic acid pathway.

In some embodiments, the non-naturally occurring microbial organismfurther includes a caprolactam pathway that includes at least oneexogenous nucleic acid encoding an enzyme selected from 1) Hydrogenase,2) Carbon monoxide dehydrogenase, 3) 3-Oxoadipyl-CoA thiolase, 4)3-Oxoadipyl-CoA reductase, 5) 3-Hydroxyadipyl-CoA dehydratase, 6)5-Carboxy-2-pentenoyl-CoA reductase, 7) Adipyl-CoA reductase (aldehydeforming), 8) 6-Aminocaproate transaminase, or 6-Aminocaproatedehydrogenase, 9) 6-Aminocaproyl-CoA/acyl-CoA transferase, or6-Aminocaproyl-CoA synthase, 10) amidohydrolase, and 11) Spontaneouscyclization.

In some embodiments, the non-naturally occurring microbial organismfurther includes a glycerol pathway that includes at least one exogenousnucleic acid encoding an enzyme selected from 1) Hydrogenase, 2) Carbonmonoxide dehydrogenase, 3) Dihydroxyacetone kinase, and 4) Glyceroldehydrogenase.

In some embodiments, the non-naturally occurring microbial organismincludes a 1,3-propanediol pathway that at least one exogenous nucleicacid encoding an enzyme selected from 1) Hydrogenase, 2) Carbon monoxidedehydrogenase, 3) Dihydroxyacetone kinase, 4) Glycerol dehydrogenase, 5)Glycerol dehydratase, and 6) 1,3-Propanediol dehydrogenase.

In some embodiments, the non-naturally occurring microbial organismincludes a microbial organism having: a reductive TCA pathway thatincludes at least one exogenous nucleic acid encoding a reductive TCApathway enzyme; the at least one exogenous nucleic acid is selected froman ATP-citrate lyase, a citrate lyase, a fumarate reductase, and analpha-ketoglutarate:ferredoxin oxidoreductase; and at least oneexogenous enzyme selected from a carbon monoxide dehydrogenase, ahydrogenase, a NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin,expressed in a sufficient amount to allow the utilization of 1) CO, 2)CO₂ and H₂, 3) CO and CO₂, 4) synthesis gas comprising CO and H₂, and 5)synthesis gas comprising CO, CO₂, and H₂.

In some embodiments, the non-naturally occurring microbial organismfurther includes at least one exogenous nucleic acid encoding a citratelyase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoAsynthetase, a succinyl-CoA transferase, a fumarase, a malatedehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoAsynthetase, and a ferredoxin.

In some embodiments, the non-naturally occurring microbial organismfurther includes an isopropanol pathway, the isopropanol pathwayconverting acetyl-CoA to isopropanol, The isopropanol pathwayincludes 1) an acetoacetyl-CoA thiolase, 2) an acetoacetyl-CoAtransferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoAsynthetase, or a phosphotransacetoacetylase/acetoacetate kinase, 3) anacetoacetate decarboxylase, and 4) an isopropanol dehydrogenase.

In some embodiments, the non-naturally occurring microbial organismincludes at least one enzyme of the isopropanol pathway that is encodedby an exogenous nucleic acid.

In some embodiments, the non-naturally occurring microbial organismincludes at least two enzymes of the isopropanol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least three enzymes of the isopropanol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes four enzymes of the isopropanol pathway which are encoded byexogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismfurther includes a 1,3-butanediol pathway, the 1,3-butanediol pathwayconverting acetyl-CoA to 1,3-butanediol. The 1,3-butanediol pathwayincludes at least three enzymes selected from 1) Acetoacetyl-CoAthiolase (AtoB), 2) Acetoacetyl-CoA reductase (CoA-dependent, alcoholforming), 3) 3-oxobutyraldehyde reductase (aldehyde reducing), 4)4-hydroxy,2-butanone reductase, 5) Acetoacetyl-CoA reductase(CoA-dependent, aldehyde forming), 6) 3-oxobutyraldehyde reductase(ketone reducing), 7) 3-hydroxybutyraldehyde reductase, 8)Acetoacetyl-CoA reductase (ketone reducing), 9) 3-hydroxybutyryl-CoAreductase (aldehyde forming), 10) 3-hydroxybutyryl-CoA reductase(alcohol forming), 11) an acetoacetyl-CoA transferase, anacetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or aphosphotransacetoacetylase/acetoacetate kinase, 12) Acetoacetatereductase, 13) 3-hydroxybutyryl-CoA transferase, hydrolase, orsynthetase, 14) 3-hydroxybutyrate reductase, and 15) 3-hydroxybutyratedehydrogenase.

In some embodiments, the non-naturally occurring microbial organismincludes at least one enzyme of the 1,3-butanediol pathway that isencoded by an exogenous nucleic acid.

In some embodiments, the non-naturally occurring microbial organismincludes at least two enzymes of the 1,3-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least three enzymes of the 1,3-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismfurther includes a 1,4-butanediol pathway, the 1,4-butanediol pathwayconverting acetyl-CoA to 1,4-butanediol. The 1,4-butanediol pathwayincludes at least five enzymes selected from 1) Acetoacetyl-CoA thiolase(AtoB), 2) 3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3) Crotonase (Crt),4) Crotonyl-CoA hydratase (4-Budh), 5) 4-hydroxybutyryl-CoA reductase(alcohol forming), 6) 4-hydroxybutyryl-CoA reductase (aldehyde forming),7) 1,4-butanediol dehydrogenase, 8) 4-Hydroxybutyryl-CoA transferase,4-Hydroxybutyryl-CoA synthetase, 4-Hydroxybutyryl-CoA hydrolase, orPhosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate kinase, and 9)4-Hydroxybutyrate reductase.

In some embodiments, the non-naturally occurring microbial organismincludes at least one enzyme of the 1,4-butanediol pathway that isencoded by an exogenous nucleic acid.

In some embodiments, the non-naturally occurring microbial organismincludes at least two enzymes of the 1,4-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least three enzymes of the 1,4-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least four enzymes of the 1,4-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least five enzymes of the 1,4-butanediol pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismfurther includes a 4-hydroxybutyrate pathway, the 4-hydroxybutyratepathway converting acetyl-CoA to 4-hydroxybutyrate. The4-hydroxybutyrate pathway includes at least five enzymes selectedfrom 1) Acetoacetyl-CoA thiolase (AtoB), 2) 3-Hydroxybutyryl-CoAdehydrogenase (Hbd), 3) Crotonase (Crt), 4) Crotonyl-CoA hydratase(4-Budh), 5) 4-Hydroxybutyryl-CoA transferase, hydrolase or synthetase,6) Phosphotrans-4-hydroxybutyrylase, and 7) 4-Hydroxybutyrate kinase.

In some embodiments, the non-naturally occurring microbial organismincludes at least one enzyme of the 4-hydroxybutyrate pathway that isencoded by an exogenous nucleic acid.

In some embodiments, the non-naturally occurring microbial organismincludes at least two enzymes of the 4-hydroxybutyrate pathway which areencoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least three enzymes of the 4-hydroxybutyrate pathway whichare encoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least four enzymes of the 4-hydroxybutyrate pathway whichare encoded by exogenous nucleic acids.

In some embodiments, the non-naturally occurring microbial organismincludes at least five enzymes of the 4-hydroxybutyrate pathway whichare encoded by exogenous nucleic acids.

Enzymes of the reductive TCA cycle useful in the non-naturally occurringmicrobial organisms of the present invention include one or more ofATP-citrate lyase and three CO₂-fixing enzymes: isocitratedehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase,pyruvate:ferredoxin oxidoreductase. The presence of ATP-citrate lyase orcitrate lyase and alpha-ketoglutarate:ferredoxin oxidoreductaseindicates the presence of an active reductive TCA cycle in an organism.Enzymes for each of these steps are shown below.

ATP citrate lyase (ACL, EC 2.3.3.8), also called ATP citrate synthase,catalyzes the ATP-dependent cleavage of citrate to oxaloacetate andacetyl-CoA. ACL is an enzyme of the RTCA cycle that has been studied ingreen sulfur bacteria Chlorobium limicola and Chlorobium tepidum. Thealpha(4)beta(4) heteromeric enzyme from Chlorobium limicola was clonedand characterized in E. coli (Kanao et al., Eur. J. Biochem.269:3409-3416 (2002). The C. limicola enzyme, encoded by aclAB, isirreversible and activity of the enzyme is regulated by the ratio ofADP/ATP. The Chlorobium tepidum a recombinant ACL from Chlorobiumtepidum was also expressed in E. coli and the holoenzyme wasreconstituted in vitro, in a study elucidating the role of the alpha andbeta subunits in the catalytic mechanism (Kim and Tabita, J. Bacteriol.188:6544-6552 (2006). ACL enzymes have also been identified inBalnearium lithotrophicum, Sulfurihydrogenibium subterraneum and othermembers of the bacterial phylum Aquificae (Hugler et al., Environ.Microbiol. 9:81-92 (2007)). This acitivy has been reported in some fungias well. Exemplary organisms include Sordaria macrospora (Nowrousian etal., Curr. Genet. 37:189-93 (2000), Aspergillus nidulans and Yarrowialipolytica (Hynes and Murray, Eukaryotic Cell, July: 1039-1048, (2010),and Aspergillus niger (Meijer et al. J. Ind. Microbiol. Biotechnol.36:1275-1280 (2009). Other candidates can be found based on sequencehomology. Information related to these enzymes is tabulated below:

Protein GenBank ID GI Number Organism aclA BAB21376.1 12407237Chlorobium limicola aclB BAB21375.1 12407235 Chlorobium limicola aclAAAM72321.1 21647054 Chlorobium tepidum aclB AAM72322.1 21647055Chlorobium tepidum aclA ABI50076.1 114054981 Balnearium lithotrophicumaclB ABI50075.1 114054980 Balnearium lithotrophicum aclA ABI50085.1114055040 Sulfurihydrogenibium subterraneum aclB ABI50084.1 114055039Sulfurihydrogenibium subterraneum aclA AAX76834.1 62199504 Sulfurimonasdenitrificans aclB AAX76835.1 62199506 Sulfurimonas denitrificans acl1XP_504787.1 50554757 Yarrowia lipolytica acl2 XP_503231.1 50551515Yarrowia lipolytica SPBC1703.07 NP_596202.1 19112994 Schizosaccharomycespombe SPAC22A12.16 NP_593246.1 19114158 Schizosaccharomyces pombe acl1CAB76165.1 7160185 Sordaria macrospora acl2 CAB76164.1 7160184 Sordariamacrospora aclA CBF86850.1 259487849 Aspergillus nidulans aclB CBF86848259487848 Aspergillus nidulans

In some organisms the conversion of citrate to oxaloacetate andacetyl-CoA proceeds through a citryl-CoA intermediate and is catalyzedby two separate enzymes, citryl-CoA synthetase (EC 6.2.1.18) andcitryl-CoA lyase (EC 4.1.3.34) (Aoshima, M., Appl. Microbiol.Biotechnol. 75:249-255 (2007). Citryl-CoA synthetase catalyzes theactivation of citrate to citryl-CoA. The Hydrogenobacter thermophilusenzyme is composed of large and small subunits encoded by ccsA and ccsB,respectively (Aoshima et al., Mol. Micrbiol. 52:751-761 (2004)). Thecitryl-CoA synthetase of Aquifex aeolicus is composed of alpha and betasubunits encoded by sucC1 and sucD1 (Hugler et al., Environ. Microbiol.9:81-92 (2007)). Citryl-CoA lyase splits citryl-CoA into oxaloacetateand acetyl-CoA. This enzyme is a homotrimer encoded by ccl inHydrogenobacter thermophilus (Aoshima et al., Mol. Microbiol. 52:763-770(2004)) and aq_150 in Aquifex aeolicus (Hugler et al., supra (2007)).The genes for this mechanism of converting citrate to oxaloacetate andcitryl-CoA have also been reported recently in Chlorobium tepidum (Eisenet al., PNAS 99(14): 9509-14 (2002).

Protein GenBank ID GI Number Organism ccsA BAD17844.1 46849514Hydrogenobacter thermophilus ccsB BAD17846.1 46849517 Hydrogenobacterthermophilus sucC1 AAC07285 2983723 Aquifex aeolicus sucD1 AAC076862984152 Aquifex aeolicus ccl BAD17841.1 46849510 Hydrogenobacterthermophilus aq_150 AAC06486 2982866 Aquifex aeolicus CT0380 NP_66128421673219 Chlorobium tepidum CT0269 NP_661173.1 21673108 Chlorobiumtepidum CT1834 AAM73055.1 21647851 Chlorobium tepidum

Oxaloacetate is converted into malate by malate dehydrogenase (EC1.1.1.37), an enzyme which functions in both the forward and reversedirection. S. cerevisiae possesses three copies of malate dehydrogenase,MDH1 (McAlister-Henn and Thompson, J. Bacteriol. 169:5157-5166 (1987),MDH2 (Minard and McAlister-Henn, Mol. Cell. Biol. 11:370-380 (1991);Gibson and McAlister-Henn, J. Biol. Chem. 278:25628-25636 (2003)), andMDH3 (Steffan and McAlister-Henn, J. Biol. Chem. 267:24708-24715(1992)), which localize to the mitochondrion, cytosol, and peroxisome,respectively. E. coli is known to have an active malate dehydrogenaseencoded by mdh.

Protein GenBank ID GI Number Organism MDH1 NP_012838 6322765Saccharomyces cerevisiae MDH2 NP_014515 116006499 Saccharomycescerevisiae MDH3 NP_010205 6320125 Saccharomyces cerevisiae MdhNP_417703.1 16131126 Escherichia coli

Fumarate hydratase (EC 4.2.1.2) catalyzes the reversible hydration offumarate to malate. The three fumarases of E. coli, encoded by fumA,fumB and fumC, are regulated under different conditions of oxygenavailability. FumB is oxygen sensitive and is active under anaerobicconditions. FumA is active under microanaerobic conditions, and FumC isactive under aerobic growth conditions (Tseng et al., J. Bacteriol.183:461-467 (2001); Woods et al., Biochim. Biophys. Acta 954:14-26(1988); Guest et al., J. Gen. Microbiol. 131:2971-2984 (1985)). S.cerevisiae contains one copy of a fumarase-encoding gene, FUM1, whoseproduct localizes to both the cytosol and mitochondrion (Sass et al., J.Biol. Chem. 278:45109-45116 (2003)). Additional fumarase enzymes arefound in Campylobacter jejuni (Smith et al., Int. J. Biochem. Cell.Biol. 31:961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch.Biochem. Biophys. 355:49-55 (1998)) and Rattus norvegicus (Kobayashi etal., J. Biochem. 89:1923-1931 (1981)). Similar enzymes with highsequence homology include fuml from Arabidopsis thaliana and fumC fromCorynebacterium glutamicum. The MmcBC fumarase from Pelotomaculumthermopropionicum is another class of fumarase with two subunits(Shimoyama et al., FEMS Microbiol. Lett. 270:207-213 (2007)).

Protein GenBank ID GI Number Organism fumA NP_416129.1 16129570Escherichia coli fumB NP_418546.1 16131948 Escherichia coli fumCNP_416128.1 16129569 Escherichia coli FUM1 NP_015061 6324993Saccharomyces cerevisiae fumC Q8NRN8.1 39931596 Corynebacteriumglutamicum fumC O69294.1 9789756 Campylobacter jejuni fumC P8412775427690 Thermus thermophilus fumH P14408.1 120605 Rattus norvegicusMmcB YP_001211906 147677691 Pelotomaculum thermopropionicum MmcCYP_001211907 147677692 Pelotomaculum thermopropionicum

Fumarate reductase catalyzes the reduction of fumarate to succinate. Thefumarate reductase of E. coli, composed of four subunits encoded byfrdABCD, is membrane-bound and active under anaerobic conditions. Theelectron donor for this reaction is menaquinone and the two protonsproduced in this reaction do not contribute to the proton gradient(Iverson et al., Science 284:1961-1966 (1999)). The yeast genome encodestwo soluble fumarate reductase isozymes encoded by FRDS1 (Enomoto etal., DNA Res. 3:263-267 (1996)) and FRDS2 (Muratsubaki et al., Arch.Biochem. Biophys. 352:175-181 (1998)), which localize to the cytosol andpromitochondrion, respectively, and are used for anaerobic growth onglucose (Arikawa et al., FEMS Microbiol. Lett. 165:111-116 (1998)).

Protein GenBank ID GI Number Organism FRDS1 P32614 418423 Saccharomycescerevisiae FRDS2 NP_012585 6322511 Saccharomyces cerevisiae frdANP_418578.1 16131979 Escherichia coli frdB NP_418577.1 16131978Escherichia coli frdC NP_418576.1 16131977 Escherichia coli frdDNP_418475.1 16131877 Escherichia coli

The ATP-dependent acylation of succinate to succinyl-CoA is catalyzed bysuccinyl-CoA synthetase (EC 6.2.1.5). The product of the LSC1 and LSC2genes of S. cerevisiae and the sucC and sucD genes of E. coli naturallyform a succinyl-CoA synthetase complex that catalyzes the formation ofsuccinyl-CoA from succinate with the concomitant consumption of one ATP,a reaction which is reversible in vivo (Buck et al., Biochemistry24:6245-6252 (1985)). These proteins are identified below:

Protein GenBank ID GI Number Organism LSC1 NP_014785 6324716Saccharomyces cerevisiae LSC2 NP_011760 6321683 Saccharomyces cerevisiaesucC NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949Escherichia coli

Alpha-ketoglutarate:ferredoxin oxidoreductase (EC 1.2.7.3), also knownas 2-oxoglutarate synthase or 2-oxoglutarate:ferredoxin oxidoreductase(OFOR), forms alpha-ketoglutarate from CO₂ and succinyl-CoA withconcurrent consumption of two reduced ferredoxin equivalents. OFOR andpyruvate:ferredoxin oxidoreductase (PFOR) are members of a diversefamily of 2-oxoacid:ferredoxin (flavodoxin) oxidoreductases whichutilize thiamine pyrophosphate, CoA and iron-sulfur clusters ascofactors and ferredoxin, flavodoxin and FAD as electron carriers (Adamset al., Archaea. Adv. Protein Chem. 48:101-180 (1996)). Enzymes in thisclass are reversible and function in the carboxylation direction inorganisms that fix carbon by the RTCA cycle such as Hydrogenobacterthermophilus, Desulfobacter hydrogenophilus and Chlorobium species(Shiba et al. 1985; Evans et al., Proc. Natl. Acad. Sci. U.S.A. 55:92934(1966); Buchanan, 1971). The two-subunit enzyme from H. thermophilusenzyme, encoded by korAB, has been cloned and expressed in E. coli (Yunet al., Biochem. Biophys. Res. Commun. 282:589-594 (2001)). A fivesubunit OFOR from the same organism with strict substrate specificityfor succinyl-CoA, encoded by forDABGE, was recently identified andexpressed in E. coli (Yun et al. 2002). The kinetics of CO₂ fixation ofboth H. thermophilus OFOR enzymes have been characterized (Yamamoto etal., Extremophiles 14:79-85 (2010)). A CO₂-fixing OFOR from Chlorobiumthiosulfatophilum has been purified and characterized but the genesencoding this enzyme have not been identified to date. Enzyme candidatesin Chlorobium species can be inferred by sequence similarity to the H.thermophilus genes. For example, the Chlorobium limicola genome encodestwo similar proteins. Acetogenic bacteria such as Moorella thermoaceticaare predicted to encode two OFOR enzymes. The enzyme encoded byMoth_0034 is predicted to function in the CO₂-assimilating direction.The genes associated with this enzyme, Moth_0034 have not beenexperimentally validated to date but can be inferred by sequencesimilarity to known OFOR enzymes.

OFOR enzymes that function in the decarboxylation direction underphysiological conditions can also catalyze the reverse reaction. TheOFOR from the thermoacidophilic archaeon Sulfolobus sp. strain 7,encoded by ST2300, has been extensively studied (Zhang et al. 1996. Aplasmid-based expression system has been developed for efficientlyexpressing this protein in E. coli (Fukuda et al., Eur. J. Biochem.268:5639-5646 (2001)) and residues involved in substrate specificitywere determined (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80(2002)). The OFOR encoded by Ape1472/Ape1473 from Aeropyrum pernix str.K1 was recently cloned into E. coli, characterized, and found to reactwith 2-oxoglutarate and a broad range of 2-oxoacids (Nishizawa et al.,FEBS Lett. 579:2319-2322 (2005)). Another exemplary OFOR is encoded byoorDABC in Helicobacter pylori (Hughes et al. 1998). An enzyme veryspecific to alpha-ketoglutarate has been reported in Thauera aromatica(Dorner and Boll, J, Bacteriol. 184 (14), 3975-83 (2002). A similarenzyme can be found in Rhodospirillum rubrum by sequence homology. A twosubunit enzyme has also been identified in Chlorobium tepidum (Eisen etal., PNAS 99(14): 9509-14 (2002)).

Protein GenBank ID GI Number Organism korA BAB21494 12583691Hydrogenobacter thermophilus korB BAB21495 12583692 Hydrogenobacterthermophilus forD BAB62132.1 14970994 Hydrogenobacter thermophilus forABAB62133.1 14970995 Hydrogenobacter thermophilus forB BAB62134.114970996 Hydrogenobacter thermophilus forG BAB62135.1 14970997Hydrogenobacter thermophilus forE BAB62136.1 14970998 Hydrogenobacterthermophilus Clim_0204 ACD89303.1 189339900 Chlorobium limicolaClim_0205 ACD89302.1 189339899 Chlorobium limicola Clim_1123 ACD90192.1189340789 Chlorobium limicola Clim_1124 ACD90193.1 189340790 Chlorobiumlimicola Moth_1984 YP_430825.1 83590816 Moorella thermoacetica Moth_1985YP_430826.1 83590817 Moorella thermoacetica Moth_0034 YP_428917.183588908 Moorella thermoacetica ST2300 NP_378302.1 15922633 Sulfolobussp. strain 7 Ape1472 BAA80470.1 5105156 Aeropyrum pernix Ape1473BAA80471.2 116062794 Aeropyrum pernix oorD AAC38210.1 2935178Helicobacter pylori oorA AAC38211.1 2935179 Helicobacter pylori oorBAAC38212.1 2935180 Helicobacter pylori oorC AAC38213.1 2935181Helicobacter pylori CT0163 NP_661069.1 21673004 Chlorobium tepidumCT0162 NP_661068.1 21673003 Chlorobium tepidum korA CAA12243.2 19571179Thauera aromatica korB CAD27440.1 19571178 Thauera aromatica Rru_A2721YP_427805.1 83594053 Rhodospirillum rubrum Rru_A2722 YP_427806.183594054 Rhodospirillum rubrum

Isocitrate dehydrogenase catalyzes the reversible decarboxylation ofisocitrate to 2-oxoglutarate coupled to the reduction of NAD(P)⁺. IDHenzymes in Saccharomyces cerevisiae and Escherichia coli are encoded byIDP 1 and icd, respectively (Haselbeck and McAlister-Henn, J. Biol.Chem. 266:2339-2345 (1991); Nimmo, H. G., Biochem. J. 234:317-2332(1986)). The reverse reaction in the reductive TCA cycle, the reductivecarboxylation of 2-oxoglutarate to isocitrate, is favored by theNADPH-dependent CO₂-fixing IDH from Chlorobium limicola and wasfunctionally expressed in E. coli (Kanao et al., Eur. J. Biochem.269:1926-1931 (2002)). A similar enzyme with 95% sequence identity isfound in the C. tepidum genome in addititon to some other candidateslisted below.

Protein GenBank ID GI Number Organism Icd ACI84720.1 209772816Escherichia coli IDP1 AAA34703.1 171749 Saccharomyces cerevisiae IdhBAC00856.1 21396513 Chlorobium limicola Icd AAM71597.1 21646271Chlorobium tepidum icd NP_952516.1 39996565 Geobacter sulfurreducens icdYP_393560. 78777245 Sulfurimonas denitrificans

In H. thermophilus the reductive carboxylation of 2-oxoglutarate iscatalyzed by two enzymes: 2-oxoglutarate carboxylase and oxalosuccinatereductase. 2-Oxoglutarate carboxylase (EC 6.4.1.7) catalyzes theATP-dependent carboxylation of alpha-ketoglutarate to oxalosuccinate(Aoshima and Igarashi, Mol. Microbiol. 62:748-759 (2006)). This enzymeis a large complex composed of two subunits. Biotinylation of the large(A) subunit is required for enzyme function (Aoshima et al., Mol.Microbiol. 51:791-798 (2004)). Oxalosuccinate reductase (EC 1.1.1.-)catalyzes the NAD-dependent conversion of oxalosuccinate toD-threo-isocitrate. The enzyme is a homodimer encoded by icd in H.thermophilus. The kinetic parameters of this enzyme indicate that theenzyme only operates in the reductive carboxylation direction in vivo,in contrast to isocitrate dehydrogenase enzymes in other organisms(Aoshima and Igarashi, J. Bacteriol. 190:2050-2055 (2008)). Based onsequence homology, gene candidates have also been found in Thiobacillusdenitrificans and Thermocrinis albus.

Protein GenBank ID GI Number Organism cfiA BAF34932.1 116234991Hydrogenobacter thermophilus cifB BAF34931.1 116234990 Hydrogenobacterthermophilus Icd BAD02487.1 38602676 Hydrogenobacter thermophilusTbd_1556 YP_315314 74317574 Thiobacillus denitrificans Tbd_1555YP_315313 74317573 Thiobacillus denitrificans Tbd_0854 YP_31461274316872 Thiobacillus denitrificans Thal_0268 YP_003473030 289548042Thermocrinis albus Thal_0267 YP_003473029 289548041 Thermocrinis albusThal_0646 YP_003473406 289548418 Thermocrinis albus

Aconitase (EC 4.2.1.3) is an iron-sulfur-containing protein catalyzingthe reversible isomerization of citrate and iso-citrate via theintermediate cis-aconitate. Two aconitase enzymes are encoded in the E.coli genome by acnA and acnB. AcnB is the main catabolic enzyme, whileAcnA is more stable and appears to be active under conditions ofoxidative or acid stress (Cunningham et al., Microbiology 143 (Pt12):3795-3805 (1997)). Two isozymes of aconitase in Salmonellatyphimurium are encoded by acnA and acnB (Horswill andEscalante-Semerena, Biochemistry 40:4703-4713 (2001)). The S. cerevisiaeaconitase, encoded by AC01, is localized to the mitochondria where itparticipates in the TCA cycle (Gangloff et al., Mol. Cell. Biol.10:3551-3561 (1990)) and the cytosol where it participates in theglyoxylate shunt (Regev-Rudzki et al., Mol. Biol. Cell. 16:4163-4171(2005)).

Protein GenBank ID GI Number Organism acnA AAC7438.1 1787531 Escherichiacoli acnB AAC73229.1 2367097 Escherichia coli acnA NP_460671.1 16765056Salmonella typhimurium acnB NP_459163.1 16763548 Salmonella typhimuriumACO1 AAA34389.1 170982 Saccharomyces cerevisiae

Pyruvate ferredoxin oxidoreductase (PFOR) catalyzes the oxidation ofpyruvate to form acetyl-CoA. The PFOR from Desulfovibrio africanus hasbeen cloned and expressed in E. coli resulting in an active recombinantenzyme that was stable for several days in the presence of oxygen(Pieulle et al., J. Bacteriol. 179:5684-5692 (1997)). Oxygen stabilityis relatively uncommon in PFORs and is believed to be conferred by a 60residue extension in the polypeptide chain of the D. africanus enzyme.Two cysteine residues in this enzyme form a disulfide bond thatprtotects it against inactivation in the form of oxygen. This disulfidebond and the stability in the presence of oxygen has been found in otherDesulfovibrio species also (Vita et al., Biochemistry, 47: 957-64(2008)). The M. thermoacetica PFOR is also well characterized (Menon andRagsdale, Biochemistry 36:8484-8494 (1997)) and was shown to have highactivity in the direction of pyruvate synthesis during autotrophicgrowth (Furdui and Ragsdale, J. Biol. Chem. 275:28494-28499 (2000)).Further, E. coli possesses an uncharacterized open reading frame, ydbK,that encodes a protein that is 51% identical to the M. thermoaceticaPFOR. Evidence for pyruvate oxidoreductase activity in E. coli has beendescribed (Blaschkowski et al., Eur. J. Biochem. 123:563-569 (1982)).PFORs have also been described in other organisms, including,Rhodobacter capsulatas (Yakunin and Hallenbeck, Biochimica et BiophysicaActa 1409 (1998) 39-49 (1998)) and Choloboum tepidum (Eisen et al., PNAS99(14): 9509-14 (2002)). The five subunit PFOR from H. thermophilus,encoded by porEDABG, was cloned into E. coli and shown to function inboth the decarboxylating and CO₂-assimilating directions (Ikeda et al.2006; Yamamoto et al., Extremophiles 14:79-85 (2010)). Homologs alsoexist in C. carboxidivorans P7. The protein sequences of these exemplaryPFOR enzymes can be identified by the following GenBank accessionnumbers. Several additional PFOR enzymes are described in the followingreview (Ragsdale, S. W., Chem. Rev. 103:2333-2346 (2003)). Finally,flavodoxin reductases (e.g., fqrB from Helicobacter pylori orCampylobacter jejuni) (St Maurice et al., J. Bacteriol. 189:4764-4773(2007)) or Rnf-type proteins (Seedorf et al., Proc. Natl. Acad. Sci.U.S.A. 105:2128-2133 (2008); and Herrmann, J. Bacteriol 190:784-791(2008)) provide a means to generate NADH or NADPH from the reducedferredoxin generated by PFOR. These proteins are identified below.

Protein GenBank ID GI Number Organism Por CAA70873.1 1770208Desulfovibrio africanus por YP_012236.1 46581428 Desulfovibrio vulgarisstr. Hildenborough Dde_3237 ABB40031.1 78220682 DesulfoVibriodesulfuricans G20 Ddes_0298 YP_002478891.1 220903579 Desulfovibriodesulfuricans subsp. desulfuricans str. ATCC 27774 Por YP_428946.183588937 Moorella thermoacetica YdbK NP_415896.1 16129339 Escherichiacoli nifJ (CT1628) NP_662511.1 21674446 Chlorobium tepidum CJE1649YP_179630.1 57238499 Campylobacter jejuni nifJ ADE85473.1 294476085Rhodobacter capsulatus porE BAA95603.1 7768912 Hydrogenobacterthermophilus porD BAA95604.1 7768913 Hydrogenobacter thermophilus porABAA95605.1 7768914 Hydrogenobacter thermophilus porB BAA95606.1 776891Hydrogenobacter thermophilus porG BAA95607.1 7768916 Hydrogenobacterthermophilus FqrB YP_001482096.1 157414840 Campylobacter jejuni HP1164NP_207955.1 15645778 Helicobacter pylori RnfC EDK33306.1 146346770Clostridium kluyveri RnfD EDK33307.1 146346771 Clostridium kluyveri RnfGEDK33308.1 146346772 Clostridium kluyveri RnfE EDK33309.1 146346773Clostridium kluyveri RnfA EDK33310.1 146346774 Clostridium kluyveri RnfBEDK33311.1 146346775 Clostridium kluyveri

The conversion of pyruvate into acetyl-CoA can be catalyzed by severalother enzymes or their combinations thereof. For example, pyruvatedehydrogenase can transform pyruvate into acetyl-CoA with theconcomitant reduction of a molecule of NAD into NADH. It is amulti-enzyme complex that catalyzes a series of partial reactions whichresults in acylating oxidative decarboxylation of pyruvate. The enzymecomprises of three subunits: the pyruvate decarboxylase (E1),dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase(E3). This enzyme is naturally present in several organisms, includingE. coli and S. cerevisiae. In the E. coli enzyme, specific residues inthe E1 component are responsible for substrate specificity (Bisswanger,H., J. Biol. Chem. 256:815-82 (1981); Bremer, J., Eur. J. Biochem.8:535-540 (1969); Gong et al., J. Biol. Chem. 275:13645-13653 (2000)).Enzyme engineering efforts have improved the E. coli PDH enzyme activityunder anaerobic conditions (Kim et al., J. Bacteriol. 190:3851-3858(2008); Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007); Zhouet al., Biotechnol. Lett. 30:335-342 (2008)). In contrast to the E. coliPDH, the B. subtilis complex is active and required for growth underanaerobic conditions (Nakano et al., J. Bacteriol. 179:6749-6755(1997)). The Klebsiella pneumoniae PDH, characterized during growth onglycerol, is also active under anaerobic conditions (5). Crystalstructures of the enzyme complex from bovine kidney (18) and the E2catalytic domain from Azotobacter vinelandii are available (4). Yetanother enzyme that can catalyze this conversion is pyruvate formatelyase. This enzyme catalyzes the conversion of pyruvate and CoA intoacetyl-CoA and formate. Pyruvate formate lyase is a common enzyme inprokaryotic organisms that is used to help modulate anaerobic redoxbalance. Exemplary enzymes can be found in Escherichia coli encoded bypflB (Knappe and Sawers, FEMS. Microbiol Rev. 6:383-398 (1990)),Lactococcus lactis (Melchiorsen et al., Appl Microbiol Biotechnol58:338-344 (2002)), and Streptococcus mutans (Takahashi-Abbe et al.,Oral. Microbiol Immunol. 18:293-297 (2003)). E. coli possesses anadditional pyruvate formate lyase, encoded by tdcE, that catalyzes theconversion of pyruvate or 2-oxobutanoate to acetyl-CoA or propionyl-CoA,respectively (Hesslinger et al., Mol. Microbiol 27:477-492 (1998)). BothpflB and tdcE from E. coli require the presence of pyruvate formatelyase activating enzyme, encoded by pflA. Further, a short proteinencoded by yfiD in E. coli can associate with and restore activity tooxygen-cleaved pyruvate formate lyase (Vey et al., Proc. Natl. Acad.Sci. U.S.A. 105:16137-16141 (2008). Note that pflA and pflB from E. coliwere expressed in S. cerevisiae as a means to increase cytosolicacetyl-CoA for butanol production as described in WO/2008/080124].Additional pyruvate formate lyase and activating enzyme candidates,encoded by pfl and act, respectively, are found in Clostridiumpasteurianum (Weidner et al., J Bacteriol. 178:2440-2444 (1996)).

Further, different enzymes can be used in combination to convertpyruvate into acetyl-CoA. For example, in S. cerevisiae, acetyl-CoA isobtained in the cytosol by first decarboxylating pyruvate to formacetaldehyde; the latter is oxidized to acetate by acetaldehydedehydrogenase and subsequently activated to form acetyl-CoA byacetyl-CoA synthetase. Acetyl-CoA synthetase is a native enzyme inseveral other organisms including E. coli (Kumari et al., J. Bacteriol.177:2878-2886 (1995)), Salmonella enterica (Starai et al., Microbiology151:3793-3801 (2005); Starai et al., J. Biol. Chem. 280:26200-26205(2005)), and Moorella thermoacetica (described already). Alternatively,acetate can be activated to form acetyl-CoA by acetate kinase andphosphotransacetylase. Acetate kinase first converts acetate intoacetyl-phosphate with the accompanying use of an ATP molecule.Acetyl-phosphate and CoA are next converted into acetyl-CoA with therelease of one phosphate by phosphotransacetylase. Both acetate kinaseand phosphotransacetlyase are well-studied enzymes in several Clostridiaand Methanosarcina thermophila.

Yet another way of converting pyruvate to acetyl-CoA is via pyruvateoxidase. Pyruvate oxidase converts pyruvate into acetate, usingubiquione as the electron acceptor. In E. coli, this activity is encodedby poxB. PoxB has similarity to pyruvate decarboxylase of S. cerevisiaeand Zymomonas mobilis. The enzyme has a thiamin pyrophosphate cofactor(Koland and Gennis, Biochemistry 21:4438-4442 (1982)); O'Brien et al.,Biochemistry 16:3105-3109 (1977); O'Brien and Gennis, J. Biol. Chem.255:3302-3307 (1980)) and a flavin adenine dinucleotide (FAD) cofactor.Acetate can then be converted into acetyl-CoA by either acetyl-CoAsynthetase or by acetate kinase and phosphotransacetylase, as describedearlier. Some of these enzymes can also catalyze the reverse reactionfrom acetyl-CoA to pyruvate.

For enzymes that use reducing equivalents in the form of NADH or NADPH,these reduced carriers can be generated by transferring electrons fromreduced ferredoxin. Two enzymes catalyze the reversible transfer ofelectrons from reduced ferredoxins to NAD(P)⁺, ferredoxin:NAD⁺oxidoreductase (EC 1.18.1.3) and ferredoxin:NADP⁺ oxidoreductase (FNR,EC 1.18.1.2). Ferredoxin:NADP⁺ oxidoreductase (FNR, EC 1.18.1.2) has anoncovalently bound FAD cofactor that facilitates the reversibletransfer of electrons from NADPH to low-potential acceptors such asferredoxins or flavodoxins (Blaschkowski et al., Eur. J. Biochem.123:563-569 (1982); Fujii et al., 1977). The Helicobacter pylori FNR,encoded by HP1164 (fqrB), is coupled to the activity ofpyruvate:ferredoxin oxidoreductase (PFOR) resulting in thepyruvate-dependent production of NADPH (St et al. 2007). An analogousenzyme is found in Campylobacter jejuni (St et al. 2007). Aferredoxin:NADP⁺ oxidoreductase enzyme is encoded in the E. coli genomeby fpr (Bianchi et al. 1993). Ferredoxin:NAD⁺ oxidoreductase utilizesreduced ferredoxin to generate NADH from NAD⁺. In several organisms,including E. coli, this enzyme is a component of multifunctionaldioxygenase enzyme complexes. The ferredoxin:NAD⁺ oxidoreductase of E.coli, encoded by hcaD, is a component of the 3-phenylproppionatedioxygenase system involved in involved in aromatic acid utilization(Diaz et al. 1998). NADH:ferredoxin reductase activity was detected incell extracts of Hydrogenobacter thermophilus strain TK-6, although agene with this activity has not yet been indicated (Yoon et al. 2006).Finally, the energy-conserving membrane-associated Rnf-type proteins(Seedorf et al., Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008);Herrmann et al., J. Bacteriol. 190:784-791 (2008)) provide a means togenerate NADH or NADPH from reduced ferredoxin. Additionalferredoxin:NAD(P)+ oxidoreductases have been annotated in Clostridiumcarboxydivorans P7.

Protein GenBank ID GI Number Organism HP1164 NP_207955.1 15645778Helicobacter pylori CJE0663 AAW35824.1 57167045 Campylobacter jejuni fprP28861.4 399486 Escherichia coli hcaD AAC75595.1 1788892 Escherichiacoli LOC100282643 NP_001149023.1 226497434 Zea mays RnfC EDK33306.1146346770 Clostridium kluyveri RnfD EDK33307.1 146346771 Clostridiumkluyveri RnfG EDK33308.1 146346772 Clostridium kluyveri RnfE EDK33309.1146346773 Clostridium kluyveri RnfA EDK33310.1 146346774 Clostridiumkluyveri RnfB EDK33311.1 146346775 Clostridium kluyveri CcarbDRAFT_2639ZP_05392639.1 255525707 Clostridium carboxidivorans P7 CcarbDRAFT_2638ZP_05392638.1 255525706 Clostridium carboxidivorans P7 CcarbDRAFT_2636ZP_05392636.1 255525704 Clostridium carboxidivorans P7 CcarbDRAFT_5060ZP_05395060.1 255528241 Clostridium carboxidivorans P7 CcarbDRAFT_2450ZP_05392450.1 255525514 Clostridium carboxidivorans P7 CcarbDRAFT_1084ZP_05391084.1 255524124 Clostridium carboxidivorans P7

Ferredoxins are small acidic proteins containing one or more iron-sulfurclusters that function as intracellular electron carriers with a lowreduction potential. Reduced ferredoxins donate electrons toFe-dependent enzymes such as ferredoxin-NADP⁺ oxidoreductase,pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxinoxidoreductase (OFOR). The H. thermophilus gene fdx1 encodes a[4Fe-4S]-type ferredoxin that is required for the reversiblecarboxylation of 2-oxoglutarate and pyruvate by OFOR and PFOR,respectively (Yamamoto et al., Extremophiles 14:79-85 (2010)). Theferredoxin associated with the Sulfolobus solfataricus2-oxoacid:ferredoxin reductase is a monomeric dicluster [3Fe-4S][4Fe-4S]type ferredoxin (Park et al. 2006). While the gene associated with thisprotein has not been fully sequenced, the N-terminal domain shares 93%homology with the zfx ferredoxin from S. acidocaldarius. The E. coligenome encodes a soluble ferredoxin of unknown physiological function,fdx. Some evidence indicates that this protein can function iniron-sulfur cluster assembly (Takahashi and Nakamura, 1999). Additionalferredoxin proteins have been characterized in Helicobacter pylori(Mukhopadhyay et al. 2003) and Campylobacter jejuni (van Vliet et al.2001). A 2Fe-2S ferredoxin from Clostridium pasteurianum has been clonedand expressed in E. coli (Fujinaga and Meyer, Biochemical andBiophysical Research Communications, 192(3): (1993)). Acetogenicbacteria such as Moorella thermoacetica, Clostridium carboxidivorans P7and Rhodospirillum rubrum are predicted to encode several ferredoxins,listed in the table below.

Protein GenBank ID GI Number Organism fdx1 BAE02673.1 68163284Hydrogenobacter thermophilus M11214.1 AAA83524.1 144806 Clostridiumpasteurianum Zfx AAY79867.1 68566938 Sulfolobus acidocalarius FdxAAC75578.1 1788874 Escherichia coli hp_0277 AAD07340.1 2313367Helicobacter pylori fdxA CAL34484.1 112359698 Campylobacter jejuniMoth_0061 ABC18400.1 83571848 Moorella thermoacetica Moth_1200ABC19514.1 83572962 Moorella thermoacetica Moth_1888 ABC20188.1 83573636Moorella thermoacetica Moth_2112 ABC20404.1 83573852 Moorellathermoacetica CcarbDRAFT_4383 ZP_05394383.1 255527515 Clostridiumcarboxidivorans P7 CcarbDRAFT_2958 ZP_05392958.1 255526034 Clostridiumcarboxidivorans P7 CcarbDRAFT_2281 ZP_05392281.1 255525342 Clostridiumcarboxidivorans P7 CcarbDRAFT_5296 ZP_05395295.1 255528511 Clostridiumcarboxidivorans P7 CcarbDRAFT_1615 ZP_05391615.1 255524662 Clostridiumcarboxidivorans P7 CcarbDRAFT_1304 ZP_05391304.1 255524347 Clostridiumcarboxidivorans P7 Rru_A2264 ABC23064.1 83576513 Rhodospirillum rubrumRru_A1916 ABC22716.1 83576165 Rhodospirillum rubrum Rru_A2026 ABC22826.183576275 Rhodospirillum rubrum

Succinyl-CoA transferase catalyzes the conversion of succinyl-CoA tosuccinate while transferring the CoA moiety to a CoA acceptor molecule.Many transferases have broad specificity and can utilize CoA acceptorsas diverse as acetate, succinate, propionate, butyrate,2-methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate, valerate,crotonate, 3-mercaptopropionate, propionate, vinylacetate, and butyrate,among others.

The conversion of succinate to succinyl-CoA can be carried by atransferase which does not require the direct consumption of an ATP orGTP. This type of reaction is common in a number of organisms. Theconversion of succinate to succinyl-CoA can also be catalyzed bysuccinyl-CoA:Acetyl-CoA transferase. The gene product of cat1 ofClostridium kluyveri has been shown to exhibit succinyl-CoA:acetyl-CoAtransferase activity (Sohling and Gottschalk, J. Bacteriol. 178:871-880(1996)). In addition, the activity is present in Trichomonas vaginalis(van Grinsven et al. 2008) and Trypanosoma brucei (Riviere et al. 2004).The succinyl-CoA:acetate CoA-transferase from Acetobacter aceti, encodedby aarC, replaces succinyl-CoA synthetase in a variant TCA cycle(Mullins et al. 2008). Similar succinyl-CoA transferase activities arealso present in Trichomonas vaginalis (van Grinsven et al. 2008),Trypanosoma brucei (Riviere et al. 2004) and Clostridium kluyveri(Sohling and Gottschalk, 1996c). The beta-ketoadipate:succinyl-CoAtransferase encoded by pcal and pcaJ in Pseudomonas putida is yetanother candidate (Kaschabek et al. 2002). The aforementioned proteinsare identified below.

Protein GenBank ID GI Number Organism cat1 P38946.1 729048 Clostridiumkluyveri TVAG_395550 XP_001330176 123975034 Trichomonas vaginalis G3Tb11.02.0290 XP_828352 71754875 Trypanosoma brucei pcaI AAN69545.124985644 Pseudomonas putida pcaJ NP_746082.1 26990657 Pseudomonas putidaaarC ACD85596.1 189233555 Acetobacter aceti

An additional exemplary transferase that converts succinate tosuccinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid issuccinyl-CoA:3:ketoacid-CoA transferase (EC 2.8.3.5). Exemplarysuccinyl-CoA:3:ketoacid-CoA transferases are present in Helicobacterpylori (Corthesy-Theulaz et al. 1997), Bacillus subtilis, and Homosapiens (Fukao et al. 2000; Tanaka et al. 2002). The aforementionedproteins are identified below.

Protein GenBank ID GI Number Organism HPAG1_0676 YP_627417 108563101Helicobacter pylori HPAG1_0677 YP_627418 108563102 Helicobacter pyloriScoA NP_391778 16080950 Bacillus subtilis ScoB NP_391777 16080949Bacillus subtilis OXCT1 NP_000427 4557817 Homo sapiens OXCT2 NP_07140311545841 Homo sapiens

Converting succinate to succinyl-CoA by succinyl-CoA:3:ketoacid-CoAtransferase requires the simultaneous conversion of a 3-ketoacyl-CoAsuch as acetoacetyl-CoA to a 3-ketoacid such as acetoacetate. Conversionof a 3-ketoacid back to a 3-ketoacyl-CoA can be catalyzed by anacetoacetyl-CoA:acetate:CoA transferase. Acetoacetyl-CoA:acetate:CoAtransferase converts acetoacetyl-CoA and acetate to acetoacetate andacetyl-CoA, or vice versa. Exemplary enzymes include the gene productsof atoAD from E. coli (Hanai et al., Appl Environ Microbiol 73:7814-7818(2007), ctfAB from C. acetobutylicum (Jojima et al., Appl MicrobiolBiotechnol 77:1219-1224 (2008), and ctfAB from Clostridiumsaccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem.71:58-68 (2007)) are shown below.

Protein GenBank ID GI Number Organism AtoA NP_416726.1 2492994Escherichia coli AtoD NP_416725.1 2492990 Escherichia coli CtfANP_149326.1 15004866 Clostridium acetobutylicum CtfB NP_149327.115004867 Clostridium acetobutylicum CtfA AAP42564.1 31075384 Clostridiumsaccharoperbutylacetonicum CtfB AAP42565.1 31075385 Clostridiumsaccharoperbutylacetonicum

Yet another possible CoA acceptor is benzylsuccinate.Succinyl-CoA:(R)-Benzylsuccinate CoA-Transferase functions as part of ananaerobic degradation pathway for toluene in organisms such as Thaueraaromatica (Leutwein and Heider, J. Bact. 183(14) 4288-4295 (2001)).Homologs can be found in Azoarcus sp. T. aromatoleum aromaticum EbN1,and Geobacter metallireducens GS-15. The aforementioned proteins areidentified below.

Protein GenBank ID GI Number Organism bbsE AAF89840 9622535 Thaueraaromatic Bbsf AAF89841 9622536 Thauera aromatic bbsE AAU45405.1 52421824Azoarcus sp. T bbsF AAU45406.1 52421825 Azoarcus sp. T bbsE YP_158075.156476486 Aromatoleum aromaticum EbN1 bbsF YP_158074.1 56476485Aromatoleum aromaticum EbN1 Gmet_1521 YP_384480.1 78222733 Geobactermetallireducens GS-15 Gmet_1522 YP_384481.1 78222734 Geobactermetallireducens GS-15

Additionally, ygfH encodes a propionyl CoA:succinate CoA transferase inE. coli (Haller et al., Biochemistry, 39(16) 4622-4629). Close homologscan be found in, for example, Citrobacter youngae ATCC 29220, Salmonellaenterica subsp. arizonae serovar, and Yersinia intermedia ATCC 29909.The aforementioned proteins are identified below.

Protein GenBank ID GI Number Organism YgfH NP_417395.1 16130821Escherichia coli str. K-12 substr. MG1655 CIT292_04485 ZP_03838384.1227334728 Citrobacter youngae ATCC 29220 SARI_04582 YP_001573497.1161506385 Salmonella enterica subsp. arizonae serovar yinte0001_14430ZP_04635364.1 238791727 Yersinia intermedia ATCC 29909

Finally, although specific gene sequences were not provided forbutyryl-CoA:acetoacetate CoA-transferase in these references, the genesFN0272 and FN0273 have been annotated as a butyrate-acetoacetateCoA-transferase (Kapatral et al., J. Bacteriol. 184(7) 2005-2018(2002)). Homologs in Fusobacterium nucleatum such as FN1857 and FN1856also likely have the desired acetoacetyl-CoA transferase activity.FN1857 and FN1856 are located adjacent to many other genes involved inlysine fermentation and are thus very likely to encode anacetoacetate:butyrate CoA transferase (Kreimeyer, et al., J. Biol. Chem.282 (10) 7191-7197 (2007)). Additional genes from Porphyrmonasgingivalis and Thermoanaerobacter tengcongensis can be identified in asimilar fashion (Kreimeyer, et al., J. Biol. Chem. 282 (10) 7191-7197(2007)). The aforementioned proteins are identified below.

Protein GenBank ID GI Number Organism FN0272 NP_603179.1 19703617Fusobacterium nucleatum FN0273 NP_603180.1 19703618 Fusobacteriumnucleatum FN1857 NP_602657.1 19705162 Fusobacterium nucleatum FN1856NP_602656.1 19705161 Fusobacterium nucleatum PG1066 NP_905281.1 34540802Porphyromonas gingivalis W83 PG1075 NP_905290.1 34540811 Porphyromonasgingivalis W83 TTE0720 NP_622378.1 20807207 Thermoanaerobactertengcongensis MB4 TTE0721 NP_622379.1 20807208 Thermoanaerobactertengcongensis MB4

Citrate lyase (EC 4.1.3.6) catalyzes a series of reactions resulting inthe cleavage of citrate to acetate and oxaloacetate. The enzyme isactive under anaerobic conditions and is composed of three subunits: anacyl-carrier protein (ACP, gamma), an ACP transferase (alpha), and aacyl lyase (beta). Enzyme activation uses covalent binding andacetylation of an unusual prosthetic group,2′-(5″-phosphoribosyl)-3-′-dephospho-CoA, which is similar in structureto acetyl-CoA. Acylation is catalyzed by CitC, a citrate lyasesynthetase. Two additional proteins, CitG and CitX, are used to convertthe apo enzyme into the active holo enzyme (Schneider et al.,Biochemistry 39:9438-9450 (2000)). Wild type E. coli does not havecitrate lyase activity; however, mutants deficient in molybdenumcofactor synthesis have an active citrate lyase (Clark, FEMS Microbiol.Lett. 55:245-249 (1990)). The E. coli enzyme is encoded by citEFD andthe citrate lyase synthetase is encoded by citC (Nilekani and SivaRaman,Biochemistry 22:4657-4663 (1983)). The Leuconostoc mesenteroides citratelyase has been cloned, characterized and expressed in E. coli (Bekal etal., J. Bacteriol. 180:647-654 (1998)). Citrate lyase enzymes have alsobeen identified in enterobacteria that utilize citrate as a carbon andenergy source, including Salmonella typhimurium and Klebsiellapneumoniae (Bott, Arch. Microbiol. 167: 78-88 (1997); Bott and Dimroth,Mol. Microbiol. 14:347-356 (1994)). The aforementioned proteins aretabulated below.

Protein GenBank ID GI Number Organism citF AAC73716.1 1786832Escherichia coli cite AAC73717.2 87081764 Escherichia coli citDAAC73718.1 1786834 Escherichia coli citC AAC73719.2 87081765 Escherichiacoli citG AAC73714.1 1786830 citX AAC73715.1 1786831 citF CAA71633.12842397 Leuconostoc mesenteroides cite CAA71632.1 2842396 Leuconostocmesenteroides citD CAA71635.1 2842395 Leuconostoc mesenteroides citCCAA71636.1 3413797 Leuconostoc mesenteroides citG CAA71634.1 2842398Leuconostoc mesenteroides citX CAA71634.1 2842398 Leuconostocmesenteroides citF NP_459613.1 16763998 Salmonella typhimurium citeAAL19573.1 16419133 Salmonella typhimurium citD NP_459064.1 16763449Salmonella typhimurium citC NP_459616.1 16764001 Salmonella typhimuriumcitG NP_459611.1 16763996 Salmonella typhimurium citX NP_459612.116763997 Salmonella typhimurium citF CAA56217.1 565619 Klebsiellapneumoniae cite CAA56216.1 565618 Klebsiella pneumoniae citD CAA56215.1565617 Klebsiella pneumoniae citC BAH66541.1 238774045 Klebsiellapneumoniae citG CAA56218.1 565620 Klebsiella pneumoniae citX AAL60463.118140907 Klebsiella pneumoniae

Acetate kinase (EC 2.7.2.1) catalyzes the reversible ATP-dependentphosphorylation of acetate to acetylphosphate. Exemplary acetate kinaseenzymes have been characterized in many organisms including E. coli,Clostridium acetobutylicum and Methanosarcina thermophila (Ingram-Smithet al., J. Bacteriol. 187:2386-2394 (2005); Fox and Roseman, J. Biol.Chem. 261:13487-13497 (1986); Winzer et al., Microbioloy 143 (Pt10):3279-3286 (1997)). Acetate kinase activity has also beendemonstrated in the gene product of E. coli purT (Marolewski et al.,Biochemistry 33:2531-2537 (1994). Some butyrate kinase enzymes (EC2.7.2.7), for example buk1 and buk2 from Clostridium acetobutylicum,also accept acetate as a substrate (Hartmanis, M. G., J. Biol. Chem.262:617-621 (1987)).

Protein GenBank ID GI Number Organism ackA NP_416799.1 16130231Escherichia coli Ack AAB18301.1 1491790 Clostridium acetobutylicum AckAAA72042.1 349834 Methanosarcina thermophila purT AAC74919.1 1788155Escherichia coli buk1 NP_349675 15896326 Clostridium acetobutylicum buk2Q97II1 20137415 Clostridium acetobutylicum

The formation of acetyl-CoA from acetylphosphate is catalyzed byphosphotransacetylase (EC 2.3.1.8). The pta gene from E. coli encodes anenzyme that reversibly converts acetyl-CoA into acetyl-phosphate(Suzuki, T., Biochim. Biophys. Acta 191:559-569 (969)). Additionalacetyltransferase enzymes have been characterized in Bacillus subtilis(Rado and Hoch, Biochim. Biophys. Acta 321:114-125 (1973), Clostridiumkluyveri (Stadtman, E., Methods Enzymol. 1:5896-599 (1955), andThermotoga maritima (Bock et al., J. Bacteriol. 181:1861-1867 (1999)).This reaction is also catalyzed by some phosphotranbutyrylase enzymes(EC 2.3.1.19) including the ptb gene products from Clostridiumacetobutylicum (Wiesenborn et al., App. Environ. Microbiol. 55:317-322(1989); Walter et al., Gene 134:107-111 (1993)). Additional ptb genesare found in butyrate-producing bacterium L2-50 (Louis et al., J.Bacteriol. 186:2099-2106 (2004) and Bacillus megaterium (Vazquez et al.,Curr. Microbiol. 42:345-349 (2001).

Protein GenBank ID GI Number Organism Pta NP_416800.1 71152910Escherichia coli Pta P39646 730415 Bacillus subtilis Pta A5N801146346896 Clostridium kluyveri Pta Q9X0L4 6685776 Thermotoga maritimaPtb NP_349676 34540484 Clostridium acetobutylicum Ptb AAR19757.138425288 butyrate-producing bacterium L2-50 Ptb CAC07932.1 10046659Bacillus megaterium

The acylation of acetate to acetyl-CoA is catalyzed by enzymes withacetyl-CoA synthetase activity. Two enzymes that catalyze this reactionare AMP-forming acetyl-CoA synthetase (EC 6.2.1.1) and ADP-formingacetyl-CoA synthetase (EC 6.2.1.13). AMP-forming acetyl-CoA synthetase(ACS) is the predominant enzyme for activation of acetate to acetyl-CoA.Exemplary ACS enzymes are found in E. coli (Brown et al., J. Gen.Microbiol. 102:327-336 (1977)), Ralstonia eutropha (Priefert andSteinbuchel, J. Bacteriol. 174:6590-6599 (1992)), Methanothermobacterthermautotrophicus (Ingram-Smith and Smith, Archaea 2:95-107 (2007)),Salmonella enterica (Gulick et al., Biochemistry 42:2866-2873 (2003))and Saccharomyces cerevisiae (Jogl and Tong, Biochemistry 43:1425-1431(2004)). ADP-forming acetyl-CoA synthetases are reversible enzymes witha generally broad substrate range (Musfeldt and Schonheit, J. Bacteriol.184:636-644 (2002)). Two isozymes of ADP-forming acetyl-CoA synthetasesare encoded in the Archaeoglobus fulgidus genome by are encoded byAF1211 and AF1983 (Musfeldt and Schonheit, supra (2002)). The enzymefrom Haloarcula marismortui (annotated as a succinyl-CoA synthetase)also accepts acetate as a substrate and reversibility of the enzyme wasdemonstrated (Brasen and Schonheit, Arch. Microbiol. 182:277-287(2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeonPyrobaculum aerophilum showed the broadest substrate range of allcharacterized ACDs, reacting with acetate, isobutyryl-CoA (preferredsubstrate) and phenylacetyl-CoA (Brasen and Schonheit, supra (2004)).Directed evolution or engineering can be used to modify this enzyme tooperate at the physiological temperature of the host organism. Theenzymes from A. fulgidus, H. marismortui and P. aerophilum have all beencloned, functionally expressed, and characterized in E. coli (Brasen andSchonheit, supra (2004); Musfeldt and Schonheit, supra (2002)).Additional candidates include the succinyl-CoA synthetase encoded bysucCD in E. coli (Buck et al., Biochemistry 24:6245-6252 (1985)) and theacyl-CoA ligase from Pseudomonas putida (Fernandez-Valverde et al.,Appl. Environ. Microbiol. 59:1149-1154 (1993)). The aforementionedproteins are tabulated below.

Protein GenBank ID GI Number Organism acs AAC77039.1 1790505 Escherichiacoli acoE AAA21945.1 141890 Ralstonia eutropha acs1 ABC87079.1 86169671Methanothermobacter thermautotrophicus acs1 AAL23099.1 16422835Salmonella enterica ACS1 Q01574.2 257050994 Saccharomyces cerevisiaeAF1211 NP_070039.1 11498810 Archaeoglobus fulgidus AF1983 NP_070807.111499565 Archaeoglobus fulgidus scs YP_135572.1 55377722 Haloarculamarismortui PAE3250 NP_560604.1 18313937 Pyrobaculum aerophilum str. IM2sucC NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949Escherichia coli paaF AAC24333.2 22711873 Pseudomonas putida

Formate dehydrogenase is a two subunit selenocysteine-containing proteinthat catalyzes the incorporation of CO₂ into formate in Moorellathermoacetica (Andreesen and Ljungdahl, J. Bacteriol. 116:867-873(1973); Li et al., J. Bacteriol. 92:4-50412 (1966); Yamamoto et al., J.Biol. Chem. 258:1826-1832 (1983)). The loci, Moth_2312 and Moth_2313 areactually one gene that is responsible for encoding the alpha subunit offormate dehydrogenase while the beta subunit is encoded by Moth_2314(Pierce et al., Environ. Microbiol. 10:2550-2573 (2008)). Another set ofgenes encoding formate dehydrogenase activity with a propensity for CO₂reduction is encoded by Sfum_2703 through Sfum_2706 in Syntrophobacterfumaroxidans (Reda et al., Proc. Natl. Acad. Sci. U.S.A. 105:10654-10658(2008); de Bok et al., Eur. J. Biochem. 270:2476-2485 (2003). Similar totheir M. thermoacetica counterparts, Sfum_2705 and Sfum_2706 areactually one gene. A similar set of genes that have been indicated tocarry out the same function are encoded by CHY 0731, CHY 0732, and CHY0733 in C. hydrogenoformans (Wu et al., PLoS Genet. 1:e65 (2005)).Homologs are also found in C. carboxidivorans P7.

Protein GenBank ID GI Number Organism Moth_2312 YP_431142 148283121Moorella thermoacetica Moth_2314 YP_431144 83591135 Moorellathermoacetica Sfum_2703 YP_846816.1 116750129 Syntrophobacterfumaroxidans Sfum_2704 YP_846817.1 116750130 Syntrophobacterfumaroxidans Sfum_2705 YP_846818.1 116750131 Syntrophobacterfumaroxidans Sfum_2706 YP_846819.1 116750132 Syntrophobacterfumaroxidans CHY_0731 YP_359585.1 78044572 Carboxydothermushydrogenoformans CHY_0732 YP_359586.1 78044500 Carboxydothermushydrogenoformans CHY_0733 YP_359587.1 78044647 Carboxydothermushydrogenoformans CcarbDRAFT_0901 ZP_05390901.1 255523938 Clostridiumcarboxidivorans P7 CcarbDRAFT_4380 ZP_05394380.1 255527512 Clostridiumcarboxidivorans P7

Formyltetrahydrofolate synthetase ligates formate to tetrahydrofolate atthe expense of one ATP. This reaction is catalyzed by the gene productof Moth_0109 in M. thermoacetica (O'brien et al., Experientia Suppl.26:249-262 (1976); Lovell et al., Arch. Microbiol. 149:280-285 (1988);Lovell et al., Biochemistry 29:5687-5694 (1990)), FHS in Clostridiumacidurici (Whitehead and Rabinowitz, J. Bacteriol. 167:203-209 (1986);Whitehead and Rabinowitz, J. Bacteriol. 170:3255-3261 (1988), andCHY_2385 in C. hydrogenoformans (Wu et al., PLoS Genet. 1:e65 (2005).Homologs exist in C. carboxidivorans P7. This enzyme is found in severalother organisms as listed below.

Protein GenBank ID GI number Organism Moth_0109 YP_428991.1 83588982Moorella thermoacetica CHY_2385 YP_361182.1 78045024 Carboxydothermushydrogenoformans FHS P13419.1 120562 Clostridium aciduriciCcarbDRAFT_1913 ZP_05391913.1 255524966 Clostridium carboxidivorans P7CcarbDRAFT_2946 ZP_05392946.1 255526022 Clostridium carboxidivorans P7Dhaf_0555 ACL18622.1 219536883 Desulfitobacterium hafniense fhsYP_001393842.1 153953077 Clostridium kluyveri DSM 555 fhs YP_003781893.1300856909 Clostridium ljungdahlii DSM 13528

In M. thermoacetica, E. coli, and C. hydrogenoformans,methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolatedehydrogenase are carried out by the bi-functional gene products ofMoth_1516, folD, and CHY_1878, respectively (Pierce et al., Environ.Microbiol. 10:2550-2573 (2008); Wu et al., PLoS Genet. 1:e65 (2005);D'Ari and Rabinowitz, J. Biol. Chem. 266:23953-23958 (1991)). A homologexists in C. carboxidivorans P7. Several other organisms also encode forthis bifunctional protein as tabulated below.

Protein GenBank ID GI number Organism Moth_1516 YP_430368.1 83590359Moorella thermoacetica folD NP_415062.1 16128513 Escherichia coliCHY_1878 YP_3 60698.1 78044829 Carboxydothermus hydrogenoformansCcarbDRAFT_2948 ZP_05392948.1 255526024 Clostridium carboxidivorans P7folD ADK16789.1 300437022 Clostridium ljungdahlii DSM 13528 folD-2NP_951919.1 39995968 Geobacter sulfurreducens PCA folD YP_725874.1113867385 Ralstonia eutropha H16 folD NP_348702.1 15895353 Clostridiumacetobutylicum ATCC 824 folD YP_696506.1 110800457 Clostridiumperfringens

The final step of the methyl branch of the Wood-Ljungdahl pathway iscatalyzed by methylenetetrahydrofolate reductase. In M. thermoacetica,this enzyme is oxygen-sensitive and contains an iron-sulfur cluster(Clark and Ljungdahl, J. Biol. Chem. 259:10845-10849 (1984). This enzymeis encoded by metF in E. coli (Sheppard et al., J. Bacteriol.181:718-725 (1999) and CHY_1233 in C. hydrogenoformans (Wu et al., PLoSGenet. 1:e65 (2005). The M. thermoacetica genes, and its C.hydrogenoformans counterpart, are located near the CODH/ACS genecluster, separated by putative hydrogenase and heterodisulfide reductasegenes. Some additional gene candidates found bioinformatically arelisted below.

Protein GenBank ID GI number Organism Moth_1191 YP_430048.1 83590039Moorella thermoacetica metF NP_418376.1 16131779 Escherichia coliCHY_1233 YP_360071.1 78044792 Carboxydothermus hydrogenoformansCLJU_c37610 YP_003781889.1 300856905 Clostridium ljungdahlii DSM 13528DesfrDRAFT_3717 ZP_07335241.1 303248996 Desulfovibrio fructosovorans JJCcarbDRAFT_2950 ZP_05392950.1 255526026 Clostridium carboxidivorans P7Ccel74_010100023124 ZP_07633513.1 307691067 Clostridium cellulovorans743B Cphy_3110 YP_001560205.1 160881237 Clostridium phytofermentans ISDg

ACS/CODH is the central enzyme of the carbonyl branch of theWood-Ljungdahl pathway. It catalyzes the reversible reduction of carbondioxide to carbon monoxide and also the synthesis of acetyl-CoA fromcarbon monoxide, Coenzyme A, and the methyl group from a methylatedcorrinoid-iron-sulfur protein. The corrinoid-iron-sulfur-protein ismethylated by methyltetrahydrofolate via a methyltransferase. Expressionof ACS/CODH in a foreign host entails introducing one or more of thefollowing proteins and their corresponding activities:Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE),Corrinoid iron-sulfur protein (AcsD), Nickel-protein assembly protein(AcsF), Ferredoxin (Orf7), Acetyl-CoA synthase (AcsB and AcsC), Carbonmonoxide dehydrogenase (AcsA), and Nickel-protein assembly protein(CooC).

The genes used for carbon-monoxide dehydrogenase/acetyl-CoA synthaseactivity typically reside in a limited region of the native genome thatcan be an extended operon (Ragsdale, S. W., Crit. Rev. Biochem. Mol.Biol. 39:165-195 (2004); Morton et al., J. Biol. Chem. 266:23824-23828(1991); Roberts et al., Proc. Natl. Acad. Sci. U.S.A. 86:32-36 (1989).Each of the genes in this operon from the acetogen, M. thermoacetica,has already been cloned and expressed actively in E. coli (Morton et al.supra; Roberts et al. supra; Lu et al., J. Biol. Chem. 268:5605-5614(1993). The protein sequences of these genes can be identified by thefollowing GenBank accession numbers.

Protein GenBank ID GI number Organism AcsE YP_430054 83590045 Moorellathermoacetica AcsD YP_430055 83590046 Moorella thermoacetica AcsFYP_430056 83590047 Moorella thermoacetica Orf7 YP_430057 83590048Moorella thermoacetica AcsC YP_430058 83590049 Moorella thermoaceticaAcsB YP_430059 83590050 Moorella thermoacetica AcsA YP_430060 83590051Moorella thermoacetica CooC YP_430061 83590052 Moorella thermoacetica

The hydrogenic bacterium, Carboxydothermus hydrogenoformans, can utilizecarbon monoxide as a growth substrate by means of acetyl-CoA synthase(Wu et al., PLoS Genet. 1:e65 (2005)). In strain Z-2901, the acetyl-CoAsynthase enzyme complex lacks carbon monoxide dehydrogenase due to aframeshift mutation (Wu et al. supra (2005)), whereas in strain DSM6008, a functional unframeshifted full-length version of this proteinhas been purified (Svetlitchnyi et al., Proc. Natl. Acad. Sci. U.S.A.101:446-451 (2004)). The protein sequences of the C. hydrogenoformansgenes from strain Z-2901 can be identified by the following GenBankaccession numbers.

Protein GenBank ID GI number Organism AcsE YP_360065 78044202Carboxydothermus hydrogenoformans AcsD YP_360064 78042962Carboxydothermus hydrogenoformans AcsF YP_360063 78044060Carboxydothermus hydrogenoformans Orf7 YP_360062 78044449Carboxydothermus hydrogenoformans AcsC YP_360061 78043584Carboxydothermus hydrogenoformans AcsB YP_360060 78042742Carboxydothermus hydrogenoformans CooC YP_360059 78044249Carboxydothermus hydrogenoformans

Homologous ACS/CODH genes can also be found in the draft genome assemblyof Clostridium carboxidivorans P7.

Protein GenBank ID GI Number Organism AcsA ZP_05392944.1 255526020Clostridium carboxidivorans P7 CooC ZP_05392945.1 255526021 Clostridiumcarboxidivorans P7 AcsF ZP_05392952.1 255526028 Clostridiumcarboxidivorans P7 AcsD ZP_05392953.1 255526029 Clostridiumcarboxidivorans P7 AcsC ZP_05392954.1 255526030 Clostridiumcarboxidivorans P7 AcsE ZP_05392955.1 255526031 Clostridiumcarboxidivorans P7 AcsB ZP_05392956.1 255526032 Clostridiumcarboxidivorans P7 Orf7 ZP_05392958.1 255526034 Clostridiumcarboxidivorans P7

The methanogenic archaeon, Methanosarcina acetivorans, can also grow oncarbon monoxide, exhibits acetyl-CoA synthase/carbon monoxidedehydrogenase activity, and produces both acetate and formate (Lessneret al., Proc. Natl. Acad. Sci. U.S.A. 103:17921-17926 (2006)). Thisorganism contains two sets of genes that encode ACS/CODH activity(Rother and Metcalf, Proc. Natl. Acad. Sci. U.S.A. 101:16929-16934(2004)). The protein sequences of both sets of M. acetivorans genes areidentified by the following GenBank accession numbers.

Protein GenBank ID GI number Organism AcsC NP_618736 20092661Methanosarcina acetivorans AcsD NP_618735 20092660 Methanosarcinaacetivorans AcsF, CooC NP_618734 20092659 Methanosarcina acetivoransAcsB NP_618733 20092658 Methanosarcina acetivorans AcsEps NP_61873220092657 Methanosarcina acetivorans AcsA NP_618731 20092656Methanosarcina acetivorans AcsC NP_615961 20089886 Methanosarcinaacetivorans AcsD NP_615962 20089887 Methanosarcina acetivorans AcsF,CooC NP_615963 20089888 Methanosarcina acetivorans AcsB NP_61596420089889 Methanosarcina acetivorans AcsEps NP_615965 20089890Methanosarcina acetivorans AcsA NP_615966 20089891 Methanosarcinaacetivorans

The AcsC, AcsD, AcsB, AcsEps, and AcsA proteins are commonly referred toas the gamma, delta, beta, epsilon, and alpha subunits of themethanogenic CODH/ACS. Homologs to the epsilon encoding genes are notpresent in acetogens such as M. thermoacetica or hydrogenogenic bacteriasuch as C. hydrogenoformans. Hypotheses for the existence of two activeCODH/ACS operons in M. acetivorans include catalytic properties (i.e.,K_(m), V_(max), k_(cat)) that favor carboxidotrophic or aceticlasticgrowth or differential gene regulation enabling various stimuli toinduce CODH/ACS expression (Rother et al., Arch. Microbiol. 188:463-472(2007)).

Expression of the modified Wood-Ljungdahl (i.e., methanol Wood-Ljungdahlpathway) in a foreign host (see FIG. 3B) requires introducing a set ofmethyltransferases to utilize the carbon and hydrogen provided bymethanol and the carbon provided by CO and/or CO₂. A complex of3-methyltransferase proteins, denoted MtaA, MtaB, and MtaC, perform thedesired methanol methyltransferase activity (Sauer et al., Eur. J.Biochem. 243:670-677 (1997); Naidu and Ragsdale, J. Bacteriol.183:3276-3281 (2001); Tallant and Krzycki, J. Biol. Chem. 276:4485-4493(2001); Tallant and Krzycki, J. Bacteriol. 179:6902-6911 (1997); Tallantand Krzycki, J. Bacteriol. 178:1295-1301 (1996); Ragsdale, S. W., Crit.Rev. Biochem. Mol. Biol. 39:165-195 (2004)).

MtaB is a zinc protein that catalyzes the transfer of a methyl groupfrom methanol to MtaC, a corrinoid protein. Exemplary genes encodingMtaB and MtaC can be found in methanogenic archaea such asMethanosarcina barkeri (Maeder et al., J. Bacteriol. 188:7922-7931(2006) and Methanosarcina acetivorans (Galagan et al., Genome Res.12:532-542 (2002), as well as the acetogen, Moorella thermoacetica (Daset al., Proteins 67:167-176 (2007). In general, the MtaB and MtaC genesare adjacent to one another on the chromosome as their activities aretightly interdependent. The protein sequences of various MtaB and MtaCencoding genes in M. barkeri, M acetivorans, and M. thermoaceticum canbe identified by their following GenBank accession numbers.

Protein GenBank ID GI number Organism MtaB1 YP_304299 73668284Methanosarcina barkeri MtaC1 YP_304298 73668283 Methanosarcina barkeriMtaB2 YP_307082 73671067 Methanosarcina barkeri MtaC2 YP_307081 73671066Methanosarcina barkeri MtaB3 YP_304612 73668597 Methanosarcina barkeriMtaC3 YP_304611 73668596 Methanosarcina barkeri MtaB1 NP_615421 20089346Methanosarcina acetivorans MtaB1 NP_615422 20089347 Methanosarcinaacetivorans MtaB2 NP_619254 20093179 Methanosarcina acetivorans MtaC2NP_619253 20093178 Methanosarcina acetivorans MtaB3 NP_616549 20090474Methanosarcina acetivorans MtaC3 NP_616550 20090475 Methanosarcinaacetivorans MtaB YP_430066 83590057 Moorella thermoacetica MtaCYP_430065 83590056 Moorella thermoacetica

The MtaB1 and MtaC1 genes, YP_304299 and YP_304298, from M. barkeri werecloned into E. coli and sequenced (Sauer et al., Eur. J. Biochem.243:670-677 (1997)). The crystal structure of this methanol-cobalaminmethyltransferase complex is also available (Hagemeier et al., Proc.Natl. Acad. Sci. U.S.A. 103:18917-18922 (2006)). The MtaB genes,YP_307082 and YP_304612, in M. barkeri were identified by sequencehomology to YP_304299. In general, homology searches are an effectivemeans of identifying methanol methyltransferases because MtaB encodinggenes show little or no similarity to methyltransferases that act onalternative substrates such as trimethylamine, dimethylamine,monomethylamine, or dimethylsulfide. The MtaC genes, YP_307081 andYP_304611, were identified based on their proximity to the MtaB genesand also their homology to YP_304298. The three sets of MtaB and MtaCgenes from M. acetivorans have been genetically, physiologically, andbiochemically characterized (Pritchett and Metcalf, Mol. Microbiol.56:1183-1194 (2005)). Mutant strains lacking two of the sets were ableto grow on methanol, whereas a strain lacking all three sets of MtaB andMtaC genes sets could not grow on methanol. This suggests that each setof genes plays a role in methanol utilization. The M. thermoacetica MtaBgene was identified based on homology to the methanogenic MtaB genes andalso by its adjacent chromosomal proximity to the methanol-inducedcorrinoid protein, MtaC, which has been crystallized (Zhou et al., ActaCrystallogr. Sect. F. Struct. Biol. Cyrst. Commun. 61:537-540 (2005) andfurther characterized by Northern hybridization and Western Blotting((Das et al., Proteins 67:167-176 (2007)).

MtaA is zinc protein that catalyzes the transfer of the methyl groupfrom MtaC to either Coenzyme M in methanogens or methyltetrahydrofolatein acetogens. MtaA can also utilize methylcobalamin as the methyl donor.Exemplary genes encoding MtaA can be found in methanogenic archaea suchas Methanosarcina barkeri (Maeder et al., J. Bacteriol. 188:7922-7931(2006) and Methanosarcina acetivorans (Galagan et al., Genome Res.12:532-542 (2002), as well as the acetogen, Moorella thermoacetica ((Daset al., Proteins 67:167-176 (2007)). In general, MtaA proteins thatcatalyze the transfer of the methyl group from CH₃-MtaC are difficult toidentify bioinformatically as they share similarity to other corrinoidprotein methyltransferases and are not oriented adjacent to the MtaB andMtaC genes on the chromosomes. Nevertheless, a number of MtaA encodinggenes have been characterized. The protein sequences of these genes inM. barkeri and M. acetivorans can be identified by the following GenBankaccession numbers.

Protein GenBank ID GI number Organism MtaA YP_304602 73668587Methanosarcina barkeri MtaA1 NP_619241 20093166 Methanosarcinaacetivorans MtaA2 NP_616548 20090473 Methanosarcina acetivorans

The MtaA gene, YP_304602, from M. barkeri was cloned, sequenced, andfunctionally overexpressed in E. coli (Harms and Thauer, Eur. J.Biochem. 235:653-659 (1996)). In M. acetivorans, MtaA1 is required forgrowth on methanol, whereas MtaA2 is dispensable even though methaneproduction from methanol is reduced in MtaA2 mutants (Bose et al., J.Bacteriol. 190:4017-4026 (2008)). It is also important to note thatthere are multiple additional MtaA homologs in M. barkeri and M.acetivorans that are as yet uncharacterized, but may also catalyzecorrinoid protein methyltransferase activity.

Putative MtaA encoding genes in M. thermoacetica were identified bytheir sequence similarity to the characterized methanogenic MtaA genes.Specifically, three M. thermoacetica genes show high homology (>30%sequence identity) to YP_304602 from M. barkeri. Unlike methanogenicMtaA proteins that naturally catalyze the transfer of the methyl groupfrom CH₃-MtaC to Coenzyme M, an M. thermoacetica MtaA is likely totransfer the methyl group to methyltetrahydrofolate given the similarroles of methyltetrahydrofolate and Coenzyme M in methanogens andacetogens, respectively. The protein sequences of putative MtaA encodinggenes from M. thermoacetica can be identified by the following GenBankaccession numbers.

Protein GenBank ID GI number Organism MtaA YP_430937 83590928 Moorellathermoacetica MtaA YP_431175 83591166 Moorella thermoacetica MtaAYP_430935 83590926 Moorella thermoacetica

Anaerobic growth on synthesis gas and methanol in the absence of anexternal electron acceptor is conferred upon the host organism with MTRand ACS/CODH activity by enabling pyruvate synthesis via pyruvateferredoxin oxidoreductase (PFOR). The gene candidates for PFOR and othermethods for converting pyruvate to acetyl-CoA are described hereinelsewhere.

The product yields per C-mol of substrate of microbial cellssynthesizing reduced fermentation products such as ethanol, butanol,isobutanol, 2-butanol, isopropanol, 1,4-butanediol, succinic acid,fumaric acid, malic acid, 4-hydroxybutyric acid, adipic acid,6-aminocaproic acid, hexamethylenediamine, caprolactam,3-hydroxyisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid,acrylic acid, 1,3-propanediol, glycerol, etc., are limited byinsufficient reducing equivalents in the carbohydrate feedstock.Reducing equivalents, or electrons, can be extracted from synthesis gascomponents such as CO and H₂ using carbon monoxide dehydrogenase (CODH)and hydrogenase enzymes, respectively. The reducing equivalents are thenpassed to acceptors such as oxidized ferredoxins, oxidized quinones,oxidized cytochromes, NADP+, water, or hydrogen peroxide to form reducedferredoxin, reduced quinones, reduced cytochromes, NAD(P)H, H₂, orwater, respectively. Reduced ferredoxin and NAD(P)H are particularlyuseful as they can serve as redox carriers for various Wood-Ljungdahlpathway and reductive TCA cycle enzymes.

Here, we show specific examples of how additional redox availabilityfrom CO and/or H₂ can improve the yields of reduced products such as1,4-BDO, 1,3-BDO, butanol, 6-aminocaproic acid, hexamethylene diamine,caprolactam, glycerol and 1,3-propanediol.

The maximum theoretical yield to produce 1,4-BDO from glucose is 1.0mole 1,4-BDO per mole of glucose under aerobic conditions via thepathways shown in FIG. 1D:

1C₆H₁₂O₆+½O₂→1C₄H₁₀O₂+2CO₂+1H₂O

Or 1.09 mol 1,4-BDO per mol of glucose under anaerobic conditions:

1C₆H₁₂O₆→1.09C₄H₁₀O₂+1.64CO₂+0.55H₂O

When both feedstocks of sugar and syngas are available, the syngascomponents CO and H₂ can be utilized to generate reducing equivalents byemploying the hydrogenase and CO dehydrogenase. The reducing equivalentsgenerated from syngas components will be utilized to power the glucoseto BDO production pathways. Theoretically, all carbons in glucose willbe conserved, thus resulting in a maximal theoretical yield to produce1,4-BDO from glucose at 2 mol 1,4-BDO per mol of glucose under eitheraerobic or anaerobic conditions as shown in FIG. 7A:

1C₆H₁₂O₆+2CO+8H₂→2C₄H₁₀O₂+4H₂O

In a similar manner, the maximum theoretical yield of 1,3-butanediol canbe improved further to 2 mol/mol glucose. An exemplary flux distributionwith the improved yields is shown in FIG. 7B.

1C₆H₁₂O₆+2CO+8H₂→2C₄H₁₀O₂+4H₂O

Butanol is yet another example of a reduced product. The production ofbutanol through fermentation has a theoretical yield of 1 mol butanolper mol of glucose. It is currently manufactured from propylene andusually used close to the point of manufacture. Butanol is largely usedas an industrial intermediate, particularly for the manufacture of butylacrylate, butyl acetate, dibutyl phthalate, dibutyl sebacate and otherbutyl esters. Other industrial uses include the manufacture ofpharmaceuticals, polymers, plastics, and herbicide. It can also be usedas a solvent for the extraction of essential oils, antibiotics,hormones, and vitamins, or as a solvent for paints, coatings, naturalresins, gums, synthetic resins, dyes, alkaloids, and camphor. Butanolhas also been proposed as the next generation biofuel to substitute fordiesel fuel and gasoline. It is also used in a wide range of consumerproducts.

1C₆H₁₂O₆→1C₄H₁₀O+2CO₂+1H₂O

When the combined feedstocks strategy is applied to butanol production,the reducing equivalents generated from syngas can increase the butanoltheoretical yield from glucose to 2 mol butanol per mol of glucose withthe pathways detailed in FIG. 7C.

1C₆H₁₂O₆+2CO+10H₂→2C₄H₁₀O+6H₂O

Hexamethylenediamine (HMDA) can be used to produce nylon 6,6, a linearpolyamide made by condensing hexamethylenediamine with adipic acid. Thisis employed for manufacturing different kinds of fibers. In addition toHMDA being used in the production of nylon-6,6, it is also utilized tomake hexamethylene diisocyanate, a monomer feedstock used in theproduction of polyurethane. The diamine also serves as a cross-linkingagent in epoxy resins. HMDA is presently produced by the hydrogenationof adiponitrile.

The production of HMDA through fermentation has a theoretical yield of0.7059 mol HMDA per mol of glucose.

17C₆H₁₂O₆+24NH₃→12C₆H₁₆N₂+30CO₂+42H₂O

When the combined feedstocks strategy is applied to the HMDA production,the reducing equivalents generated from syngas can increase the HMDAtheoretical yield from glucose to 1 mol HMDA per mol of glucose with thepathways detailed in FIG. 7D.

1C₆H₁₂O₆+2NH₃+5H₂→1C₆H₁₆N₂+6H₂O

or

1C₆H₁₂O₆+2NH₃+5CO→1C₆H₁₆N₂+H₂O+5CO₂

or

1C₆H₁₂O₆+2NH₃+2CO+3H₂→1C₆H₁₆N₂+4H₂O+2CO₂

Caprolactam is an organic compound which is a lactam of 6-aminohexanoicacid (ε-aminohexanoic acid, 6-aminocaproic acid). It can alternativelybe considered a cyclic amide of caproic acid. One use of caprolactam isas a monomer in the production of nylon-6. Caprolactam can besynthesized from cyclohexanone via an oximation process usinghydroxylammonium sulfate followed by catalytic rearrangement using theBeckmann rearrangement process step. The production of caprolactamthrough fermentation has a theoretical yield of 0.8 mol caprolactam permol of glucose.

5C₆H₁₂O₆+4NH₃→4C₆H₁₃NO₂+6CO₂+10H₂O

When the combined feedstocks strategy is applied to caprolactamproduction, the reducing equivalents generated from syngas can increasethe caprolactam theoretical yield from glucose to 1 mol caprolactam permol of glucose with the pathways detailed in FIG. 7D.

1C₆H₁₂O₆+1NH₃+3H₂→1C₆H₁₃NO₂+4H₂O

or

1C₆H₁₂O₆+1NH₃+3CO→1C₆H₁₃NO₂+1H₂O+3CO₂

or

1C₆H₁₂O₆+1NH₃+1CO+2H₂→1C₆H₁₃NO₂+3H₂O+1CO₂

Other exemplary products for which the yields on carbohydrates can beimproved by providing additional reducing equivalents are1,3-propanediol (1,3-PDO) and glycerol. 1,3-PDO is mainly used as abuilding block in the production of polymers. It can be formulated intoa variety of industrial products including composites, adhesives,laminates, coatings, moldings, aliphatic polyesters, copolyesters. It isalso a solvent and used as an antifreeze and wood paint.

1,3-PDO can be chemically synthesized via the hydration of acrolein orby the hydroformylation of ethylene oxide to afford3-hydroxypropionaldehyde. The resultant aldehyde is hydrogenated to give1,3-PDO. Additionally, 1,3-PDO can be produced biologically. Theproduction of 1,3-PDO through fermentation has a theoretical yield of1.5 mol 1,3-PDO per mol of glucose.

2C₆H₁₂O₆→3C₃H₈O₂+3CO₂

When the combined feedstock strategy is applied to 1,3-PDO production,the reducing equivalents generated from syngas can increase the 1,3-PDOtheoretical yield based on glucose to 2 mol 1,3-PDO per mol of glucoseby the pathways shown in FIG. 7E.

1C₆H₁₂O₆+4H₂→2C₃H₈O₂+2H₂O

or

1C₆H₁₂O₆+4CO+2H₂O→2C₃H₈O₂+4CO₂

or

1C₆H₁₂O₆+2CO+2H₂→2C₃H₈O₂+2CO₂

Similarly, the production of glycerol through fermentation can beimproved by the combined feedstock strategy. The production of glycerolthrough fermentation has a theoretical yield of 1.71 mol glycerol permol of glucose.

7C₆H₁₂O₆+6H₂O→12C₃H₈O₃+6CO₂

When the combined feedstocks strategy is applied to glycerol production,the reducing equivalents generated from syngas can increase the glyceroltheoretical yield from glucose to 2 mol glycerol per mol of glucose withthe pathways detailed in FIG. 7E.

1C₆H₁₂O₆+2H₂→2C₃H₈O₃

or

1C₆H₁₂O₆+2CO+2H₂O→2C₃H₈O₃+2CO₂

or

1C₆H₁₂O₆+1CO+1H₂+1H₂O→2C₃H₈O₃+1CO₂

As shown in above three examples, a combined feedstock strategy wheresyngas is combined with a sugar-based feedstock or other carbonsubstrate can greatly improve the theoretical yields. In this co-feedingapproach, syngas components H₂ and CO can be utilized by the hydrogenaseand CO dehydrogenase to generate reducing equivalents, that can be usedto power chemical production pathways in which the carbons from sugar orother carbon substrates will be maximally conserved and the theoreticalyields improved. In case of 1,4-BDO, 1,3-BDO and butanol productionsfrom glucose or sugar, the theoretical yields improve from 1 mol or 1.09mol products per mol of glucose to 2 mol products per mol of glucose.Such improvements provide environmental and economic benefits andgreatly enhance sustainable chemical production.

Herein below the enzymes and the corresponding genes used for extractingredox from synags components are described. CODH is a reversible enzymethat interconverts CO and CO₂ at the expense or gain of electrons. Thenatural physiological role of the CODH in ACS/CODH complexes is toconvert CO₂ to CO for incorporation into acetyl-CoA by acetyl-CoAsynthase. Nevertheless, such CODH enzymes are suitable for theextraction of reducing equivalents from CO due to the reversible natureof such enzymes. Expressing such CODH enzymes in the absence of ACSallows them to operate in the direction opposite to their naturalphysiological role (i.e., CO oxidation).

In M. thermoacetica, C. hydrogenoformans, C. carboxidivorans P7, andseveral other organisms, additional CODH encoding genes are locatedoutside of the ACS/CODH operons. These enzymes provide a means forextracting electrons (or reducing equivalents) from the conversion ofcarbon monoxide to carbon dioxide. The M. thermoacetica gene (GenbankAccession Number: YP_430813) is expressed by itself in an operon and isbelieved to transfer electrons from CO to an external mediator likeferredoxin in a “Ping-pong” reaction. The reduced mediator then couplesto other reduced nicolinamide adenine dinucleotide phosphate (NAD(P)H)carriers or ferredoxin-dependent cellular processes (Ragsdale, Annals ofthe New York Academy of Sciences 1125: 129-136 (2008)). The genesencoding the C. hydrogenoformans CODH-II and CooF, a neighboringprotein, were cloned and sequenced (Gonzalez and Robb, FEMS MicrobiolLett. 191:243-247 (2000)). The resulting complex was membrane-bound,although cytoplasmic fractions of CODH-II were shown to catalyze theformation of NADPH suggesting an anabolic role (Svetlitchnyi et al., JBacteriol. 183:5134-5144 (2001)). The crystal structure of the CODH-IIis also available (Dobbek et al., Science 293:1281-1285 (2001)). SimilarACS-free CODH enzymes can be found in a diverse array of organismsincluding Geobacter metallireducens GS-15, Chlorobium phaeobacteroidesDSM 266, Clostridium cellulolyticum H10, Desulfovibrio desulfuricanssubsp. desulfuricans str. ATCC 27774, Pelobacter carbinolicus DSM 2380,and Campylobacter curvus 525.92.

Protein GenBank ID GI Number Organism CODH YP_430813 83590804 Moorellathermoacetica (putative) CODH-II YP_358957 78044574 Carboxydothermushydrogenoformans (CooS-II) CooF YP_358958 78045112 Carboxydothermushydrogenoformans CODH ZP_05390164.1 255523193 Clostridiumcarboxidivorans P7 (putative) ZP_05390341.1 255523371 Clostridiumcarboxidivorans P7 ZP_05391756.1 255524806 Clostridium carboxidivoransP7 ZP_05392944.1 255526020 Clostridium carboxidivorans P7 CODHYP_384856.1 78223109 Geobacter metallireducens GS-15 Cpha266_0148YP_910642.1 119355998 Chlorobium phaeobacteroides DSM 266 (cytochrome c)Cpha266_0149 YP_910643.1 119355999 Chlorobium phaeobacteroides DSM 266(CODH) Ccel_0438 YP_002504800.1 220927891 Clostridium cellulolyticum H10Ddes_0382 YP_002478973.1 220903661 Desulfovibrio desulfuricans subsp.(CODH) desulfuricans str. ATCC 27774 Ddes_0381 YP_002478972.1 220903660Desulfovibrio desulfuricans subsp. (CooC) desulfuricans str. ATCC 27774Pcar_0057 YP_355490.1 7791767 Pelobacter carbinolicus DSM 2380 (CODH)Pcar_0058 YP_355491.1 7791766 Pelobacter carbinolicus DSM 2380 (CooC)Pcar_0058 YP_355492.1 7791765 Pelobacter carbinolicus DSM 2380 (HypA)CooS (CODH) YP_001407343.1 154175407 Campylobacter curvus 525.92

In some cases, hydrogenase encoding genes are located adjacent to aCODH. In Rhodospirillum rubrum, the encoded CODH/hydrogenase proteinsform a membrane-bound enzyme complex that has been indicated to be asite where energy, in the form of a proton gradient, is generated fromthe conversion of CO and H₂O to CO₂ and H₂ (Fox et al., J Bacteriol.178:6200-6208 (1996)). The CODH-I of C. hydrogenoformans and itsadjacent genes have been proposed to catalyze a similar functional rolebased on their similarity to the R. rubrum CODH/hydrogenase gene cluster(Wu et al., PLoS Genet. 1:e65 (2005)). The C. hydrogenoformans CODH-Iwas also shown to exhibit intense CO oxidation and CO₂ reductionactivities when linked to an electrode (Parkin et al., J Am. Chem. Soc.129:10328-10329 (2007)). The protein sequences of exemplary CODH andhydrogenase genes can be identified by the following GenBank accessionnumbers.

Protein GenBank ID GI Number Organism CODH-I (CooS- YP_360644 78043475Carboxydothermus I) hydrogenoformans CooF YP_360645 78044791Carboxydothermus hydrogenoformans HypA YP_360646 78044340Carboxydothermus hydrogenoformans CooH YP_360647 78043871Carboxydothermus hydrogenoformans CooU YP_360648 78044023Carboxydothermus hydrogenoformans CooX YP_360649 78043124Carboxydothermus hydrogenoformans CooL YP_360650 78043938Carboxydothermus hydrogenoformans CooK YP_360651 78044700Carboxydothermus hydrogenoformans CooM YP_360652 78043942Carboxydothermus hydrogenoformans CooC YP_360654.1 78043296Carboxydothermus hydrogenoformans CooA-1 YP_360655.1 78044021Carboxydothermus hydrogenoformans CooL AAC45118 1515468 Rhodospirillumrubrum CooX AAC45119 1515469 Rhodospirillum rubrum CooU AAC45120 1515470Rhodospirillum rubrum CooH AAC45121 1498746 Rhodospirillum rubrum CooFAAC45122 1498747 Rhodospirillum rubrum CODH (CooS) AAC45123 1498748Rhodospirillum rubrum CooC AAC45124 1498749 Rhodospirillum rubrum CooTAAC45125 1498750 Rhodospirillum rubrum CooJ AAC45126 1498751Rhodospirillum rubrum

Native to E. coli and other enteric bacteria are multiple genes encodingup to four hydrogenases (Sawers, G., Antonie Van Leeuwenhoek 66:57-88(1994); Sawers et al., J Bacteriol. 164:1324-1331 (1985); Sawers andBoxer, Eur. J Biochem. 156:265-275 (1986); Sawers et al., J Bacteriol.168:398-404 (1986)). Given the multiplicity of enzyme activities, E.coli or another host organism can provide sufficient hydrogenaseactivity to split incoming molecular hydrogen and reduce thecorresponding acceptor. E. coli possesses two uptake hydrogenases, Hyd-1and Hyd-2, encoded by the hyaABCDEF and hybOABCDEFG gene clusters,respectively (Lukey et al., How E. coli is equipped to oxidize hydrogenunder different redox conditions, J Biol Chem published online Nov. 16,2009). Hyd-1 is oxygen-tolerant, irreversible, and is coupled to quinonereduction via the hyaC cytochrome. Hyd-2 is sensitive to O₂, reversible,and transfers electrons to the periplasmic ferredoxin hybA which, inturn, reduces a quinone via the hybB integral membrane protein. Reducedquinones can serve as the source of electrons for fumarate reductase inthe reductive branch of the TCA cycle. Reduced ferredoxins can be usedby enzymes such as NAD(P)H:ferredoxin oxidoreductases to generate NADPHor NADH. They can alternatively be used as the electron donor forreactions such as pyruvate ferredoxin oxidoreductase, AKG ferredoxinoxidoreductase, and 5,10-methylene-H₄folate reductase.

Protein GenBank ID GI Number Organism HyaA AAC74057.1 1787206Escherichia coli HyaB AAC74058.1 1787207 Escherichia coli HyaCAAC74059.1 1787208 Escherichia coli HyaD AAC74060.1 1787209 Escherichiacoli HyaE AAC74061.1 1787210 Escherichia coli HyaF AAC74062.1 1787211Escherichia coli HybO AAC76033.1 1789371 Escherichia coli HybAAAC76032.1 1789370 Escherichia coli HybB AAC76031.1 2367183 Escherichiacoli HybC AAC76030.1 1789368 Escherichia coli HybD AAC76029.1 1789367Escherichia coli HybE AAC76028.1 1789366 Escherichia coli HybFAAC76027.1 1789365 Escherichia coli HybG AAC76026.1 1789364 Escherichiacoli

The hydrogen-lyase systems of E. coli include hydrogenase 3, amembrane-bound enzyme complex using ferredoxin as an acceptor, andhydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3 and 4are encoded by the hyc and hyf gene clusters, respectively. Hydrogenase3 has been shown to be a reversible enzyme (Maeda et al., Appl MicrobiolBiotechnol 76(5):1035-42 (2007)). Hydrogenase activity in E. coli isalso dependent upon the expression of the hyp genes whose correspondingproteins are involved in the assembly of the hydrogenase complexes(Jacobi et al., Arch. Microbiol 158:444-451 (1992); Rangaraj an et al.,J. Bacteriol. 190:1447-1458 (2008)).

Protein GenBank ID GI Number Organism HycA NP_417205 16130632Escherichia coli HycB NP_417204 16130631 Escherichia coli HycC NP_41720316130630 Escherichia coli HycD NP_417202 16130629 Escherichia coli HycENP_417201 16130628 Escherichia coli HycF NP_417200 16130627 Escherichiacoli HycG NP_417199 16130626 Escherichia coli HycH NP_417198 16130625Escherichia coli HycI NP_417197 16130624 Escherichia coli HyfA NP_41697690111444 Escherichia coli HyfB NP_416977 16130407 Escherichia coli HyfCNP_416978 90111445 Escherichia coli HyfD NP_416979 16130409 Escherichiacoli HyfE NP_416980 16130410 Escherichia coli HyfF NP_416981 16130411Escherichia coli HyfG NP_416982 16130412 Escherichia coli HyfH NP_41698316130413 Escherichia coli HyfI NP_416984 16130414 Escherichia coli HyfJNP_416985 90111446 Escherichia coli HyfR NP_416986 90111447 Escherichiacoli HypA NP_417206 16130633 Escherichia coli HypB NP_417207 16130634Escherichia coli HypC NP_417208 16130635 Escherichia coli HypD NP_41720916130636 Escherichia coli HypE NP_417210 226524740 Escherichia coli HypFNP_417192 16130619 Escherichia coli

The M. thermoacetica hydrogenases are suitable for a host that lackssufficient endogenous hydrogenase activity. M. thermoacetica can growwith CO₂ as the exclusive carbon source indicating that reducingequivalents are extracted from H₂ to enable acetyl-CoA synthesis via theWood-Ljungdahl pathway (Drake, H. L., J. Bacteriol. 150:702-709 (1982);Drake and Daniel, Res. Microbiol. 155:869-883 (2004); Kellum and Drake,J. Bacteriol. 160:466-469 (1984)) (see FIG. 2A). M. thermoacetica hashomologs to several hyp, hyc, and hyf genes from E. coli. The proteinsequences encoded for by these genes are identified by the followingGenBank accession numbers.

Proteins in M. thermoacetica whose genes are homologous to the E. colihyp genes are shown below.

Protein GenBank ID GI Number Organism Moth_2175 YP_431007 83590998Moorella thermoacetica Moth_2176 YP_431008 83590999 Moorellathermoacetica Moth_2177 YP_431009 83591000 Moorella thermoaceticaMoth_2178 YP_431010 83591001 Moorella thermoacetica Moth_2179 YP_43101183591002 Moorella thermoacetica Moth_2180 YP_431012 83591003 Moorellathermoacetica Moth_2181 YP_431013 83591004 Moorella thermoacetica

Proteins in M. thermoacetica that are homologous to the E. coliHydrogenase 3 and/or 4 proteins are listed in the following table.

Protein GenBank ID GI Number Organism Moth_2182 YP_431014 83591005Moorella thermoacetica Moth_2183 YP_431015 83591006 Moorellathermoacetica Moth_2184 YP_431016 83591007 Moorella thermoaceticaMoth_2185 YP_431017 83591008 Moorella thermoacetica Moth_2186 YP_43101883591009 Moorella thermoacetica Moth_2187 YP_431019 83591010 Moorellathermoacetica Moth_2188 YP_431020 83591011 Moorella thermoaceticaMoth_2189 YP_431021 83591012 Moorella thermoacetica Moth_2190 YP_43102283591013 Moorella thermoacetica Moth_2191 YP_431023 83591014 Moorellathermoacetica Moth_2192 YP_431024 83591015 Moorella thermoacetica

In addition, several gene clusters encoding hydrogenase functionalityare present in M. thermoacetica and their corresponding proteinsequences are provided below.

Protein GenBank ID GI Number Organism Moth_0439 YP_429313 83589304Moorella thermoacetica Moth_0440 YP_429314 83589305 Moorellathermoacetica Moth_0441 YP_429315 83589306 Moorella thermoaceticaMoth_0442 YP_429316 83589307 Moorella thermoacetica Moth_0809 YP_42967083589661 Moorella thermoacetica Moth_0810 YP_429671 83589662 Moorellathermoacetica Moth_0811 YP_429672 83589663 Moorella thermoaceticaMoth_0812 YP_429673 83589664 Moorella thermoacetica Moth_0814 YP_42967483589665 Moorella thermoacetica Moth_0815 YP_429675 83589666 Moorellathermoacetica Moth_0816 YP_429676 83589667 Moorella thermoaceticaMoth_1193 YP_430050 83590041 Moorella thermoacetica Moth_1194 YP_43005183590042 Moorella thermoacetica Moth_1195 YP_430052 83590043 Moorellathermoacetica Moth_1196 YP_430053 83590044 Moorella thermoaceticaMoth_1717 YP_430562 83590553 Moorella thermoacetica Moth_1718 YP_43056383590554 Moorella thermoacetica Moth_1719 YP_430564 83590555 Moorellathermoacetica Moth_1883 YP_430726 83590717 Moorella thermoaceticaMoth_1884 YP_430727 83590718 Moorella thermoacetica Moth_1885 YP_43072883590719 Moorella thermoacetica Moth_1886 YP_430729 83590720 Moorellathermoacetica Moth_1887 YP_430730 83590721 Moorella thermoaceticaMoth_1888 YP_430731 83590722 Moorella thermoacetica Moth_1452 YP_43030583590296 Moorella thermoacetica Moth_1453 YP_430306 83590297 Moorellathermoacetica Moth_1454 YP_430307 83590298 Moorella thermoacetica

Ralstonia eutropha H16 uses hydrogen as an energy source with oxygen asa terminal electron acceptor. Its membrane-bound uptake[NiFe]-hydrogenase is an “O2-tolerant” hydrogenase (Cracknell, et al.Proc Nat Acad Sci, 106(49) 20681-20686 (2009)) that isperiplasmically-oriented and connected to the respiratory chain via ab-type cytochrome (Schink and Schlegel, Biochim. Biophys. Acta, 567,315-324 (1979); Bernhard et al., Eur. J. Biochem. 248, 179-186 (1997)).R. eutropha also contains an O2-tolerant soluble hydrogenase encoded bythe Hox operon which is cytoplasmic and directly reduces NAD+ at theexpense of hydrogen (Schneider and Schlegel, Biochim. Biophys. Acta 452,66-80 (1976); Burgdorf, J. Bact. 187(9) 3122-3132(2005)). Solublehydrogenase enzymes are additionally present in several other organismsincluding Geobacter sulfurreducens (Coppi, Microbiology 151, 1239-1254(2005)), Synechocystis str. PCC 6803 (Germer, J. Biol. Chem., 284(52),36462-36472 (2009)), and Thiocapsa roseopersicina (Rakhely, Appl.Environ. Microbiol. 70(2) 722-728 (2004)). The Synechocystis enzyme iscapable of generating NADPH from hydrogen. Overexpression of both theHox operon from Synechocystis str. PCC 6803 and the accessory genesencoded by the Hyp operon from Nostoc sp. PCC 7120 led to increasedhydrogenase activity compared to expression of the Hox genes alone(Germer, J. Biol. Chem. 284(52), 36462-36472 (2009)).

Protein GenBank ID GI Number Organism HoxF NP_942727.1 38637753Ralstonia eutropha H16 HoxU NP_942728.1 38637754 Ralstonia eutropha H16HoxY NP_942729.1 38637755 Ralstonia eutropha H16 HoxH NP_942730.138637756 Ralstonia eutropha H16 HoxW NP_942731.1 38637757 Ralstoniaeutropha H16 HoxI NP_942732.1 38637758 Ralstonia eutropha H16 HoxENP_953767.1 39997816 Geobacter sulfurreducens HoxF NP_953766.1 39997815Geobacter sulfurreducens HoxU NP_953765.1 39997814 Geobactersulfurreducens HoxY NP_953764.1 39997813 Geobacter sulfurreducens HoxHNP_953763.1 39997812 Geobacter sulfurreducens GSU2717 NP_953762.139997811 Geobacter sulfurreducens HoxE NP_441418.1 16330690Synechocystis str. PCC 6803 HoxF NP_441417.1 16330689 Synechocystis str.PCC 6803 Unknown NP_441416.1 16330688 Synechocystis str. PCC function6803 HoxU NP_441415.1 16330687 Synechocystis str. PCC 6803 HoxYNP_441414.1 16330686 Synechocystis str. PCC 6803 Unknown NP_441413.116330685 Synechocystis str. PCC function 6803 Unknown NP_441412.116330684 Synechocystis str. PCC function 6803 HoxH NP_441411.1 16330683Synechocystis str. PCC 6803 HypF NP_484737.1 17228189 Nostoc sp. PCC7120 HypC NP_484738.1 17228190 Nostoc sp. PCC 7120 HypD NP_484739.117228191 Nostoc sp. PCC 7120 Unknown NP_484740.1 17228192 Nostoc sp. PCC7120 function HypE NP_484741.1 17228193 Nostoc sp. PCC 7120 HypANP_484742.1 17228194 Nostoc sp. PCC 7120 HypB NP_484743.1 17228195Nostoc sp. PCC 7120 Hox1E AAP50519.1 37787351 Thiocapsa roseopersicinaHox1F AAP50520.1 37787352 Thiocapsa roseopersicina Hox1U AAP5052E137787353 Thiocapsa roseopersicina Hox1Y AAP50522.1 37787354 Thiocapsaroseopersicina Hox1H AAP50523.1 37787355 Thiocapsa roseopersicina

Several enzymes and the corresponding genes used for fixing carbondioxide to either pyruvate or phosphoenolpyruvate to form the TCA cycleintermediates, oxaloacetate or malate are described below.

Carboxylation of phosphoenolpyruvate to oxaloacetate is catalyzed byphosphoenolpyruvate carboxylase. Exemplary PEP carboxylase enzymes areencoded by ppc in E. coli (Kai et al., Arch. Biochem. Biophys.414:170-179 (2003), ppcA in Methylobacterium extorquens AM1 (Arps etal., J. Bacteriol. 175:3776-3783 (1993), and ppc in Corynebacteriumglutamicum (Eikmanns et al., Mol. Gen. Genet. 218:330-339 (1989).

Protein GenBank ID GI Number Organism Ppc NP_418391 16131794 Escherichiacoli ppcA AAB58883 28572162 Methylobacterium extorquens Ppc ABB5327080973080 Corynebacterium glutamicum

An alternative enzyme for converting phosphoenolpyruvate to oxaloacetateis PEP carboxykinase, which simultaneously forms an ATP whilecarboxylating PEP. In most organisms PEP carboxykinase serves agluconeogenic function and converts oxaloacetate to PEP at the expenseof one ATP. S. cerevisiae is one such organism whose native PEPcarboxykinase, PCK1, serves a gluconeogenic role (Valdes-Hevia et al.,FEBS Lett. 258:313-316 (1989). E. coli is another such organism, as therole of PEP carboxykinase in producing oxaloacetate is believed to beminor when compared to PEP carboxylase, which does not form ATP,possibly due to the higher K_(m) for bicarbonate of PEP carboxykinase(Kim et al., Appl. Environ. Microbiol. 70:1238-1241 (2004)).Nevertheless, activity of the native E. coli PEP carboxykinase from PEPtowards oxaloacetate has been recently demonstrated in ppc mutants of E.coli K-12 (Kwon et al., J. Microbiol. Biotechnol. 16:1448-1452 (2006)).These strains exhibited no growth defects and had increased succinateproduction at high NaHCO₃ concentrations. Mutant strains of E. coli canadopt Pck as the dominant CO₂-fixing enzyme following adaptive evolution(Zhang et al. 2009). In some organisms, particularly rumen bacteria, PEPcarboxykinase is quite efficient in producing oxaloacetate from PEP andgenerating ATP. Examples of PEP carboxykinase genes that have beencloned into E. coli include those from Mannheimia succiniciproducens(Lee et al., Biotechnol. Bioprocess Eng. 7:95-99 (2002)),Anaerobiospirillum succiniciproducens (Laivenieks et al., Appl. Environ.Microbiol. 63:2273-2280 (1997), and Actinobacillus succinogenes (Kim etal. supra). The PEP carboxykinase enzyme encoded by Haemophilusinfluenza is effective at forming oxaloacetate from PEP.

Protein GenBank ID GI Number Organism PCK1 NP_013023  6322950Saccharomyces cerevisiae pck NP_417862.1 16131280 Escherichia coli pckAYP_089485.1 52426348 Mannheimia succiniciproducens pckA O09460.1 3122621 Anaerobiospirillum succiniciproducens pckA Q6W6X5 75440571Actinobacillus succinogenes pckA P43923.1  1172573 Haemophilus influenza

Pyruvate carboxylase (EC 6.4.1.1) directly converts pyruvate tooxaloacetate at the cost of one ATP. Pyruvate carboxylase enzymes areencoded by PYC1 (Walker et al., Biochem. Biophys. Res. Commun.176:1210-1217 (1991) and PYC2 (Walker et al., supra) in Saccharomycescerevisiae, and pyc in Mycobacterium smegmatis (Mukhopadhyay andPurwantini, Biochim. Biophys. Acta 1475:191-206 (2000)).

Protein GenBank ID GI Number Organism PYC1 NP_011453  6321376Saccharomyces cerevisiae PYC2 NP_009777  6319695 Saccharomycescerevisiae Pyc YP_890857.1 118470447 Mycobacterium smegmatis

Malic enzyme can be applied to convert CO₂ and pyruvate to malate at theexpense of one reducing equivalent. Malic enzymes for this purpose caninclude, without limitation, malic enzyme (NAD-dependent) and malicenzyme (NADP-dependent). For example, one of the E. coli malic enzymes(Takeo, J. Biochem. 66:379-387 (1969)) or a similar enzyme with higheractivity can be expressed to enable the conversion of pyruvate and CO₂to malate. By fixing carbon to pyruvate as opposed to PEP, malic enzymeallows the high-energy phosphate bond from PEP to be conserved bypyruvate kinase whereby ATP is generated in the formation of pyruvate orby the phosphotransferase system for glucose transport. Although malicenzyme is typically assumed to operate in the direction of pyruvateformation from malate, overexpression of the NAD-dependent enzyme,encoded by maeA, has been demonstrated to increase succinate productionin E. coli while restoring the lethal Δpfl-ΔldhA phenotype underanaerobic conditions by operating in the carbon-fixing direction (Stolsand Donnelly, Appl. Environ. Microbiol. 63(7) 2695-2701 (1997)). Asimilar observation was made upon overexpressing the malic enzyme fromAscaris suum in E. coli (Stols et al., Appl. Biochem. Biotechnol.63-65(1), 153-158 (1997)). The second E. coli malic enzyme, encoded bymaeB, is NADP-dependent and also decarboxylates oxaloacetate and otheralpha-keto acids (Iwakura et al., J. Biochem. 85(5):1355-65 (1979)).

Protein GenBank ID GI Number Organism maeA NP_415996 90111281Escherichia coli maeB NP_416958 16130388 Escherichia coli NAD-ME P27443 126732 Ascaris suum

The enzymes used for converting oxaloacetate (formed from, for example,PEP carboxylase, PEP carboxykinase, or pyruvate carboxylase) or malate(formed from, for example, malic enzyme or malate dehydrogenase) tosuccinyl-CoA via the reductive branch of the TCA cycle are malatedehydrogenase, fumarate dehydratase (fumarase), fumarate reductase, andsuccinyl-CoA transferase. The genes for each of the enzymes aredescribed herein above.

Enzymes, genes and methods for engineering pathways from succinyl-CoA tovarious products into a microorganism are now known in the art. Theadditional reducing equivalents obtained from CO and H₂, as disclosedherein, improve the yields of all these products when utilizingcarbohydrate-based feedstock. For example, 1,4-butanediol can beproduced from succinyl-CoA via previously disclosed pathways (see forexample, Burk et al., WO 2008/115840). Exemplary enzymes for theconversion succinyl-CoA to 1,4-butanediol include succinyl-CoA reductase(aldehyde forming), 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyratekinase, phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoA reductase(aldehyde forming), 1,4-butanediol dehydrogenase, succinyl-CoA reductase(alcohol forming), 4-hydroxybutyryl-CoA transferase,4-hydroxybutyryl-CoA synthetase, 4-hydroxybutyryl-phosphate reductase,4-hydroxybutyrate reductase, and 4-hydroxybutyryl-CoA reductase (alcoholforming). Succinate reductase can be additionally useful in convertingsuccinate directly to the 1,4-butanediol pathway intermediate, succinatesemialdehyde. Finally, succinyl-CoA can be converted byalpha-ketoglutarate:ferredoxin oxidoreductase to alpha-ketoglutaratewhose decarboxylation by alpha-ketoglutarate decarboxylase leads to theformation of succinate semialdehyde.

1,3-butanediol can be produced from succinyl-CoA via the pathways havebeen described. Exemplary enzymes for the conversion succinyl-CoA to1,3-butanediol include succinyl-CoA reductase (aldehyde forming),4-hydroxybutyrate dehydrogenase, 4-hydroxybutyrate kinase,phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoA dehydratase,crotonase, 3-hydroxybutyryl-CoA reductase (aldehyde forming),3-hydroxybutyraldehyde reductase, succinyl-CoA reductase (alcoholforming), 4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoAsynthetase, 3-hydroxybutyryl-CoA hydrolase, 3-hydroxybutyryl-CoAsynthetase, 3-hydroxybutyryl-CoA transferase, 3-hydroxybutyratereductase, 3-hydroxybutyryl-CoA reductase (alcohol forming). Succinatereductase can be additionally useful by converting succinate directly tothe 1,3-butanediol pathway intermediate, succinate semialdehyde.Finally, succinyl-CoA can be converted by alpha-ketoglutarate:ferredoxinoxidoreductase to alpha-ketoglutarate whose decarboxylation byalpha-ketoglutarate decarboxylase leads to the formation of succinatesemialdehyde.

n-butanol can be produced from succinyl-CoA via known pathways.Exemplary enzymes for the conversion succinyl-CoA to butanol includesuccinyl-CoA reductase (aldehyde forming), 4-hydroxybutyratedehydrogenase, 4-hydroxybutyrate kinase,phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoA dehydratase,butyryl-CoA dehydrogenase, butyryl-CoA hydrolase, butyryl-coAsynthetase, butyryl-coA transferase, butyrate reductase, butyryl-CoAreductase (aldehyde forming), butyraldehyde reductase, butyryl-CoAreductase (alcohol forming), succinyl-CoA reductase (alcohol forming),4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA synthetase.Succinate reductase can be additionally useful by converting succinatedirectly to the butanol pathway intermediate, succinate semialdehyde.Finally, succinyl-CoA can be converted by alpha-ketoglutarate:ferredoxinoxidoreductase to alpha-ketoglutarate whose decarboxylation byalpha-ketoglutarate decarboxylase leads to the formation of succinatesemialdehyde.

Isobutanol can be produced from succinyl-CoA via known pathways.Exemplary enzymes for the conversion succinyl-CoA to isobutanol includesuccinyl-CoA reductase (aldehyde forming), 4-hydroxybutyratedehydrogenase, 4-hydroxybutyrate kinase,phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoA dehydratase,butyryl-CoA dehydrogenase, isobutyryl-CoA mutase, isobutyryl-CoAhydrolase, isobutyryl-coA synthetase, isobutyryl-coA transferase,isobutyrate reductase, isobutyryl-CoA reductase (aldehyde forming),isobutyraldehyde reductase, isobutyryl-CoA reductase (alcohol forming),succinyl-CoA reductase (alcohol forming), 4-hydroxybutyryl-CoAtransferase, 4-hydroxybutyryl-CoA synthetase. Succinate reductase can beadditionally useful by converting succinate directly to the isobutanolpathway intermediate, succinate semialdehyde. Finally, succinyl-CoA canbe converted by alpha-ketoglutarate:ferredoxin oxidoreductase toalpha-ketoglutarate whose decarboxylation by alpha-ketoglutaratedecarboxylase leads to the formation of succinate semialdehyde.

Isopropanol can be produced from succinyl-CoA via known pathways.Exemplary enzymes for the conversion succinyl-CoA to isopropanol includesuccinyl-CoA reductase (aldehyde forming), 4-hydroxybutyratedehydrogenase, 4-hydroxybutyrate kinase,phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoA dehydratase,crotonase, 3-hydroxybutyryl-CoA dehydrogenase, acetoacetyl-CoAsynthetase, acetoacetate-CoA transferase, acetoacetyl-CoA hydrolase,acetoacetate decarboxylase, acetone reductase, succinyl-CoA reductase(alcohol forming), 4-hydroxybutyryl-CoA transferase, and4-hydroxybutyryl-CoA synthetase. Succinate reductase can be additionallyuseful by converting succinate directly to the isopropanol pathwayintermediate, succinate semialdehyde. Finally, succinyl-CoA can beconverted by alpha-ketoglutarate:ferredoxin oxidoreductase toalpha-ketoglutarate whose decarboxylation by alpha-ketoglutaratedecarboxylase leads to the formation of succinate semialdehyde.

n-propanol can be produced from succinyl-CoA via known pathways.Exemplary enzymes for the conversion succinyl-CoA to n-propanol includepropionaldehyde dehydrogenase, propanol dehydrogenase,propionyl-CoA:phosphate propanoyltransferase, propionyl-CoA hydrolase,propionyl-CoA transferase, propionyl-CoA synthetase, propionate kinase,propionate reductase, propionyl phosphate reductase, methylmalonyl-CoAmutase, methylmalonyl-CoA epimerase, methylmalonyl-CoA decarboxylase,and methylmalonyl-CoA carboxytransferase.

Adipate can be produced from succinyl-CoA via known pathways (see forexample, Burgard et al., (WO/2009/151728A2). Exemplary enzymes for theconversion of succinyl-CoA to adipate include succinyl-CoA:acetyl-CoAacyl transferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoAdehydratase, 5-carboxy-2-pentenoyl-CoA reductase, adipyl-CoA synthetase,phosphotransadipylase, adipate kinase, adipyl-CoA:acetyl-CoAtransferase, adipyl-CoA hydrolase, 3-oxoadipyl-CoA transferase,3-oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoatereductase.

6-aminocaproate can be produced from succinyl-CoA via known pathways.Exemplary enzymes for the conversion of succinyl-CoA to 6-aminocaproateinclude succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoAdehydrogenase, 3-hydroxyadipyl-CoA dehydratase,5-carboxy-2-pentenoyl-CoA reductase, adipyl-CoA synthetase,phosphotransadipylase, adipate kinase, adipyl-CoA:acetyl-CoAtransferase, adipyl-CoA hydrolase, adipate reductase, adipyl-CoAreductase, CoA-dependent aldehyde dehydrogenase (e.g., adipyl-CoAreductase (aldehyde forming), transaminase (e.g., 6-aminocaproatetransaminase), 6-aminocaproate dehydrogenase, 3-oxoadipyl-CoAtransferase, 3-oxoadipate reductase, 3-hydroxyadipate dehydratase, and2-enoate reductase.

Hexamethylenediamine can be produced from succinyl-CoA via knownpathways. Exemplary enzymes for the conversion of succinyl-CoA toadipate include succinyl-CoA:acetyl-CoA acyl transferase,3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase,5-carboxy-2-pentenoyl-CoA reductase, adipyl-CoA synthetase,phosphotransadipylase, adipate kinase, adipyl-CoA:acetyl-CoAtransferase, adipyl-CoA hydrolase, adipate reductase, adipyl-CoAreductase, CoA-dependent aldehyde dehydrogenase (e.g., adipyl-CoAreductase (aldehyde forming), transaminase (e.g., 6-aminocaproatetransaminase), 6-aminocaproate dehydrogenase, 3-oxoadipyl-CoAtransferase, 3-oxoadipate reductase, 3-hydroxyadipate dehydratase,2-enoate reductase, 6-aminocaproyl-CoA/acyl-CoA transferase,6-aminocaproyl-CoA synthase, 6-aminocaproyl-CoA reductase (aldehydeforming), hexamethylenediamine transaminase, and hexamethylenediaminedehydrogenase.

Enzymes, genes and methods for engineering pathways from glycolysisintermediates to various products into a microorganism are known in theart. The additional reducing equivalents obtained from CO and H₂, asdescribed herein, improve the yields of all these products oncarbohydrates. For example, glycerol and 1,3-propanediol can be producedfrom the glycolysis intermediate, dihydroxyacetone phosphate, via thepathways described in (Nakamura and Whited, Curr. Opin. Biotechnol.14(5) 454-459 (2003)). Exemplary enzymes for the conversion ofdihydroxyacetone phosphate to glycerol include glycerol-3-phosphatedehydrogenase and glycerol-3-phosphate phosphatase. Exemplary enzymesfor the conversion of dihydroxyacetone phosphate to 1,3-propanediolinclude glycerol-3-phosphate dehydrogenase, glycerol-3-phosphatephosphatase, glycerol dehydratase, and 1,3-propanediol oxidoreductase.

In some embodiments, the reductive TCA cycle, coupled with carbonmonoxide and hydrogenase enzymes, can be employed to allow syngasutilization by microorganisms. Synthesis gas (syngas) is a mixture ofprimarily H₂ and CO that can be obtained via gasification of any organicfeedstock, such as coal, coal oil, natural gas, biomass, or wasteorganic matter. Numerous gasification processes have been developed, andmost designs are based on partial oxidation, where limiting oxygenavoids full combustion, of organic materials at high temperatures(500-1500° C.) to provide syngas as a 0.5:1-3:1 H₂/CO mixture. Inaddition to coal, biomass of many types has been used for syngasproduction and represents an inexpensive and flexible feedstock for thebiological production of renewable chemicals and fuels.

The CO₂-fixing reductive tricarboxylic acid (RTCA) cycle is anendergenic anabolic pathway of CO₂ assimilation, requiring reducingequivalents and ATP. The reductive TCA cycle was first reported in thegreen sulfur photosynthetic bacterium Chlorobium limicola (Evans et al.,Proc. Natl. Acad. Sci. U.S.A. 55:928-934 (1966)). Similar pathways havebeen characterized in some prokaryotes (proteobacteria, green sulfurbacteria and thermophillic Knallgas bacteria) and sulfur-dependentarchaea (Hugler et al., J. Bacteriol. 187:3020-3027 (2005; Hugler etal., Environ. Microbiol. 9:81-92 (2007). In some cases, reductive andoxidative (Krebs) TCA cycles are present in the same organism (Hugler etal., supra (2007); Siebers et al., J. Bacteriol. 186:2179-2194 (2004)).Some methanogens and obligate anaerobes possess incomplete oxidative orreductive TCA cycles that may function to synthesize biosyntheticintermediates (Ekiel et al., J. Bacteriol. 162:905-908 (1985); Wood etal., FEMS Microbiol. Rev. 28:335-352 (2004)).

The components of synthesis gas can provide sufficient CO₂, reducingequivalents, and ATP for the reductive TCA cycle to operate. One turn ofthe RTCA cycle assimilates two moles of CO₂ into one mole of acetyl-CoAand requires 2 ATP and 4 reducing equivalents. CO and H₂ can providereducing equivalents by means of carbon monoxide dehydrogenase andhydrogenase enzymes, respectively. Reducing equivalents can come in theform of NADH, NADPH, FADH, reduced quinones, reduced ferredoxins, andreduced flavodoxins. The reducing equivalents, particularly NADH, NADPH,and reduced ferredoxin, can serve as cofactors for the RTCA cycleenzymes (e.g., malate dehydrogenase, fumarate reductase,alpha-ketoglutarate:ferredoxin oxidoreductase (alternatively known as2-oxoglutarate:ferredoxin oxidoreductase, alpha-ketoglutarate synthase,or 2-oxoglutarate synthase), and isocitrate dehydrogenase). Theelectrons from these reducing equivalents can alternatively pass throughan ion-gradient producing electron transport chain where they are passedto an acceptor such as oxygen, nitrate, oxidized metal ions, protons, oran electrode. The ion-gradient can then be used for ATP generation viaan ATP synthase or similar enzyme.

Many of the enzymes in the TCA cycle are reversible and can catalyzereactions in the reductive and oxidative directions. However, some TCAcycle reactions are irreversible in vivo and thus different enzymes areused to catalyze these reactions in the directions required for thereverse TCA cycle. These reactions are: 1. conversion of citrate tooxaloacetate and acetyl-CoA, 2. conversion of fumarate to succinate, 3.conversion of succinyl-CoA to alpha-ketoglutarate. In the TCA cycle,citrate is formed from the condensation of oxaloacetate and acetyl-CoA.The reverse reaction, cleavage of citrate to oxaloacetate andacetyl-CoA, is ATP-dependent and catalyzed by ATP citrate lyase orcitryl-CoA synthetase and citryl-CoA lyase. Alternatively, citrate lyasecan be coupled to acetyl-CoA synthetase, an acetyl-CoA transferase, orphosphotransacetylase and acetate kinase to form acetyl-CoA andoxaloacetate from citrate. The conversion of succinate to fumarate iscatalyzed by succinate dehydrogenase while the reverse reaction iscatalyzed by fumarate reductase. In the TCA cycle succinyl-CoA is formedfrom the NAD(P)⁺ dependent decarboxylation of oxaloacetate by thealpha-ketoglutarate dehydrogenase complex. The reverse reaction iscatalyzed by alpha-ketoglutarate:ferredoxin oxidoreductase.

An organism capable of utilizing the reverse tricarboxylic acid cycle toenable production of acetyl-CoA-derived products on 1) CO, 2) CO₂ andH₂, 3) CO and CO₂, 4) synthesis gas comprising CO and H₂, and 5)synthesis gas comprising CO, CO₂, and H₂ can include any of thefollowing enzyme activities: ATP-citrate lyase, citrate lyase,aconitase, isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxinoxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase,fumarate reductase, fumarase, malate dehydrogenase, acetate kinase,phosphotransacetylase, acetyl-CoA synthetase, NAD(P)H:ferredoxinoxidoreductase, carbon monoxide dehydrogenase, hydrogenase, andferredoxin (see FIG. 8). Enzyme enzymes and the corresponding genesrequired for these activities are described herein above.

Carbon from syngas can be fixed via the reverse TCA cycle and componentsthereof. Specifically, the combination of certain syngas-utilizationpathway components with the pathways for formation of isopropanol,butanol, 4-hydroxybutyrate, 1,3-butanediol, or 1,4-butanediol fromacetyl-CoA results in high yields of these products by providing anefficient mechanism for fixing the carbon present in carbon dioxide, fedexogenously or produced endogenously from CO, into acetyl-CoA (seebelow).

3CO+6H₂→C₃H₈O+2H₂O

9CO+4H₂O→C₃H₈O+6CO₂

9H₂+3CO₂→C₃H₈O+5H₂O  Isopropanol:

4CO+8H₂→C₄H₁₀O+3H₂O

12CO+5H₂O→C₄H₁₀O+8CO₂

12H₂+4CO₂→C₄H₁₀O+5H₂O  Butanol:

4CO+5H₂→C₄H₈O₃+H₂O

9CO+4H₂O→C₄H₈O₃+5CO₂

9H₂+4CO₂→C₄H₈O₃+5H₂O  4-Hydroxybutyrate:

4CO+7H₂→C₄H₁₀O₂+2H₂O

11CO+5H₂O→C₄H₁₀O₂+7CO₂

11H₂+4CO₂→C₄H₁₀O₂+6H₂O  1,3 or 1,4-butanediol:

The organisms and conversion routes described herein provide anefficient means of converting synthesis gas and its components toproducts such as isopropanol, butanol, 4-hydroxybutyrate, 1,3-butanediolor 1,4-butanediol. Additional product molecules that can be produced bythe teachings of this invention include but are not limited to ethanol,n-propanol, isobutanol, succinic acid, fumaric acid, malic acid,3-hydroxypropionic acid, lactic acid, adipic acid, 6-aminocaproic acid,hexamethylenediamine, 3-hydoxyisobutyric acid, 2-hydroxyisobutyric acid,methacrylic acid, acrylic acid, and long chain hydrocarbons, alcohols,acids, and esters.

While generally described herein as a microbial organism that contains a1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, isopropanol pathway,or other product, it is understood that the invention additionallyprovides a non-naturally occurring microbial organism comprising atleast one exogenous nucleic acid encoding a 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol pathway enzymeexpressed in a sufficient amount to produce an intermediate of a1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolpathway, or other product intermediate. For example, as disclosedherein, a 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol pathway are exemplified in FIGS. 1-4. Therefore, in additionto a microbial organism containing a 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol pathway that produces 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol, the inventionadditionally provides a non-naturally occurring microbial organismcomprising at least one exogenous nucleic acid encoding a1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolpathway enzyme, where the microbial organism produces a 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol pathway intermediate.

It is understood that any of the pathways disclosed herein, as describedin the Examples and exemplified in the Figures, including the pathwaysof FIGS. 1-8, can be utilized to generate a non-naturally occurringmicrobial organism that produces any pathway intermediate or product, asdesired. As disclosed herein, such a microbial organism that produces anintermediate can be used in combination with another microbial organismexpressing downstream pathway enzymes to produce a desired product.However, it is understood that a non-naturally occurring microbialorganism that produces a 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol pathway intermediate can be utilized toproduce the intermediate as a desired product.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing, or a protein associated with, thereferenced metabolic reaction, reactant or product. Unless otherwiseexpressly stated herein, those skilled in the art will understand thatreference to a reaction also constitutes reference to the reactants andproducts of the reaction. Similarly, unless otherwise expressly statedherein, reference to a reactant or product also references the reaction,and reference to any of these metabolic constituents also references thegene or genes encoding the enzymes that catalyze or proteins involved inthe referenced reaction, reactant or product. Likewise, given the wellknown fields of metabolic biochemistry, enzymology and genomics,reference herein to a gene or encoding nucleic acid also constitutes areference to the corresponding encoded enzyme and the reaction itcatalyzes or a protein associated with the reaction as well as thereactants and products of the reaction.

The non-naturally occurring microbial organisms of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes or proteins participating in one or more 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol biosynthetic pathways.Depending on the host microbial organism chosen for biosynthesis,nucleic acids for some or all of a particular 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol biosynthetic pathwaycan be expressed. For example, if a chosen host is deficient in one ormore enzymes or proteins for a desired biosynthetic pathway, thenexpressible nucleic acids for the deficient enzyme(s) or protein(s) areintroduced into the host for subsequent exogenous expression.Alternatively, if the chosen host exhibits endogenous expression of somepathway genes, but is deficient in others, then an encoding nucleic acidis needed for the deficient enzyme(s) or protein(s) to achieve1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolbiosynthesis. Thus, a non-naturally occurring microbial organism of theinvention can be produced by introducing exogenous enzyme or proteinactivities to obtain a desired biosynthetic pathway or a desiredbiosynthetic pathway can be obtained by introducing one or moreexogenous enzyme or protein activities that, together with one or moreendogenous enzymes or proteins, produces a desired product such as1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanol.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus or any of a variety of other microorganisms applicable tofermentation processes. Exemplary bacteria include species selected fromEscherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacteriumglutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcuslactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.Exemplary yeasts or fungi include species selected from Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichiapastoris, Rhizopus arrhizus, Rhizopus oryzae, and the like. E. coli is aparticularly useful host organism since it is a well characterizedmicrobial organism suitable for genetic engineering. Other particularlyuseful host organisms include yeast such as Saccharomyces cerevisiae. Itis understood that any suitable microbial host organism can be used tointroduce metabolic and/or genetic modifications to produce a desiredproduct.

Depending on the 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol biosynthetic pathway constituents of a selected hostmicrobial organism, the non-naturally occurring microbial organisms ofthe invention will include at least one exogenously expressed1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolpathway-encoding nucleic acid and up to all encoding nucleic acids forone or more 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol biosynthetic pathways. For example, 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol biosynthesis can beestablished in a host deficient in a pathway enzyme or protein throughexogenous expression of the corresponding encoding nucleic acid. In ahost deficient in all enzymes or proteins of a 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol pathway, exogenousexpression of all enzyme or proteins in the pathway can be included,although it is understood that all enzymes or proteins of a pathway canbe expressed even if the host contains at least one of the pathwayenzymes or proteins. For example, exogenous expression of all enzymes orproteins in a pathway for production of 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol can be included.

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel the1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolpathway deficiencies of the selected host microbial organism. Therefore,a non-naturally occurring microbial organism of the invention can haveone, two, three, four, five, six, seven, eight, or up to all nucleicacids encoding the enzymes or proteins constituting a 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol biosynthetic pathwaydisclosed herein. In some embodiments, the non-naturally occurringmicrobial organisms also can include other genetic modifications thatfacilitate or optimize 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol biosynthesis or that confer other usefulfunctions onto the host microbial organism. One such other functionalitycan include, for example, augmentation of the synthesis of one or moreof the 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolpathway precursors.

Generally, a host microbial organism is selected such that it producesthe precursor of a 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol pathway, either as a naturally produced molecule or as anengineered product that either provides de novo production of a desiredprecursor or increased production of a precursor naturally produced bythe host microbial organism. A host organism can be engineered toincrease production of a precursor, as disclosed herein. In addition, amicrobial organism that has been engineered to produce a desiredprecursor can be used as a host organism and further engineered toexpress enzymes or proteins of a 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol pathway.

In some embodiments, a non-naturally occurring microbial organism of theinvention is generated from a host that contains the enzymaticcapability to synthesize 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol. In this specific embodiment it can beuseful to increase the synthesis or accumulation of a 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol pathway product to,for example, drive 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol pathway reactions toward 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol production. Increased synthesis oraccumulation can be accomplished by, for example, overexpression ofnucleic acids encoding one or more of the above-described1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolpathway enzymes or proteins. Over expression the enzyme or enzymesand/or protein or proteins of the 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol pathway can occur, for example, throughexogenous expression of the endogenous gene or genes, or throughexogenous expression of the heterologous gene or genes. Therefore,naturally occurring organisms can be readily generated to benon-naturally occurring microbial organisms of the invention, forexample, producing 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol, through overexpression of one, two, three, four, five, six,seven, eight, up to all nucleic acids encoding 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol biosynthetic pathwayenzymes or proteins. In addition, a non-naturally occurring organism canbe generated by mutagenesis of an endogenous gene that results in anincrease in activity of an enzyme in the 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol biosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism.

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, a 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol biosynthetic pathway onto the microbial organism.Alternatively, encoding nucleic acids can be introduced to produce anintermediate microbial organism having the biosynthetic capability tocatalyze some of the required reactions to confer 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol biosyntheticcapability. For example, a non-naturally occurring microbial organismhaving a 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol biosynthetic pathway can comprise at least two exogenousnucleic acids encoding desired enzymes or proteins. Thus, it isunderstood that any combination of two or more enzymes or proteins of abiosynthetic pathway can be included in a non-naturally occurringmicrobial organism of the invention. Similarly, it is understood thatany combination of three or more enzymes or proteins of a biosyntheticpathway can be included in a non-naturally occurring microbial organismof the invention, as desired, so long as the combination of enzymesand/or proteins of the desired biosynthetic pathway results inproduction of the corresponding desired product. Similarly, anycombination of four or more enzymes or proteins of a biosyntheticpathway as disclosed herein can be included in a non-naturally occurringmicrobial organism of the invention, as desired, so long as thecombination of enzymes and/or proteins of the desired biosyntheticpathway results in production of the corresponding desired product.

In addition to the biosynthesis of 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol as described herein, the non-naturallyoccurring microbial organisms and methods of the invention also can beutilized in various combinations with each other and with othermicrobial organisms and methods well known in the art to achieve productbiosynthesis by other routes. For example, one alternative to produce1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanol otherthan use of the 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol producers is through addition of another microbial organismcapable of converting a 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol pathway intermediate to 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol. One such procedureincludes, for example, the fermentation of a microbial organism thatproduces a 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol pathway intermediate. The 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol pathway intermediate can then be used asa substrate for a second microbial organism that converts the1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolpathway intermediate to 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol. The 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol pathway intermediate can be addeddirectly to another culture of the second organism or the originalculture of the 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol pathway intermediate producers can be depleted of thesemicrobial organisms by, for example, cell separation, and thensubsequent addition of the second organism to the fermentation broth canbe utilized to produce the final product without intermediatepurification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol. In these embodiments,biosynthetic pathways for a desired product of the invention can besegregated into different microbial organisms, and the differentmicrobial organisms can be co-cultured to produce the final product. Insuch a biosynthetic scheme, the product of one microbial organism is thesubstrate for a second microbial organism until the final product issynthesized. For example, the biosynthesis of 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol can be accomplished byconstructing a microbial organism that contains biosynthetic pathwaysfor conversion of one pathway intermediate to another pathwayintermediate or the product. Alternatively, 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol also can bebiosynthetically produced from microbial organisms through co-culture orco-fermentation using two organisms in the same vessel, where the firstmicrobial organism produces a 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol intermediate and the second microbialorganism converts the intermediate to 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methods ofthe invention together with other microbial organisms, with theco-culture of other non-naturally occurring microbial organisms havingsubpathways and with combinations of other chemical and/or biochemicalprocedures well known in the art to produce 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol.

Sources of encoding nucleic acids for a 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol pathway enzyme orprotein can include, for example, any species where the encoded geneproduct is capable of catalyzing the referenced reaction. Such speciesinclude both prokaryotic and eukaryotic organisms including, but notlimited to, bacteria, including archaea and eubacteria, and eukaryotes,including yeast, plant, insect, animal, and mammal, including human.Exemplary species for such sources include, for example, Escherichiacoli, S. cerevisiae, B. subtilis, Candida boidinii, as well as otherexemplary species disclosed herein or available as source organisms forcorresponding genes. However, with the complete genome sequenceavailable for now more than 550 species (with more than half of theseavailable on public databases such as the NCBI), including 395microorganism genomes and a variety of yeast, fungi, plant, andmammalian genomes, the identification of genes encoding the requisite1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolbiosynthetic activity for one or more genes in related or distantspecies, including for example, homologues, orthologs, paralogs andnonorthologous gene displacements of known genes, and the interchange ofgenetic alterations between organisms is routine and well known in theart. Accordingly, the metabolic alterations allowing biosynthesis of1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanoldescribed herein with reference to a particular organism such as E. colican be readily applied to other microorganisms, including prokaryoticand eukaryotic organisms alike. Given the teachings and guidanceprovided herein, those skilled in the art will know that a metabolicalteration exemplified in one organism can be applied equally to otherorganisms.

In some instances, such as when an alternative 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol biosynthetic pathwayexists in an unrelated species, 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol biosynthesis can be conferred onto thehost species by, for example, exogenous expression of a paralog orparalogs from the unrelated species that catalyzes a similar, yetnon-identical metabolic reaction to replace the referenced reaction.Because certain differences among metabolic networks exist betweendifferent organisms, those skilled in the art will understand that theactual gene usage between different organisms may differ. However, giventhe teachings and guidance provided herein, those skilled in the artalso will understand that the teachings and methods of the invention canbe applied to all microbial organisms using the cognate metabolicalterations to those exemplified herein to construct a microbialorganism in a species of interest that will synthesize 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol.

Methods for constructing and testing the expression levels of anon-naturally occurring 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol-producing host can be performed, forexample, by recombinant and detection methods well known in the art.Such methods can be found described in, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring HarborLaboratory, New York (2001); and Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for production of1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanol can beintroduced stably or transiently into a host cell using techniques wellknown in the art including, but not limited to, conjugation,electroporation, chemical transformation, transduction, transfection,and ultrasound transformation. For exogenous expression in E. coli orother prokaryotic cells, some nucleic acid sequences in the genes orcDNAs of eukaryotic nucleic acids can encode targeting signals such asan N-terminal mitochondrial or other targeting signal, which can beremoved before transformation into prokaryotic host cells, if desired.For example, removal of a mitochondrial leader sequence led to increasedexpression in E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338(2005)). For exogenous expression in yeast or other eukaryotic cells,genes can be expressed in the cytosol without the addition of leadersequence, or can be targeted to mitochondrion or other organelles, ortargeted for secretion, by the addition of a suitable targeting sequencesuch as a mitochondrial targeting or secretion signal suitable for thehost cells. Thus, it is understood that appropriate modifications to anucleic acid sequence to remove or include a targeting sequence can beincorporated into an exogenous nucleic acid sequence to impart desirableproperties. Furthermore, genes can be subjected to codon optimizationwith techniques well known in the art to achieve optimized expression ofthe proteins.

An expression vector or vectors can be constructed to include one ormore 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolbiosynthetic pathway encoding nucleic acids as exemplified hereinoperably linked to expression control sequences functional in the hostorganism. Expression vectors applicable for use in the microbial hostorganisms of the invention include, for example, plasmids, phagevectors, viral vectors, episomes and artificial chromosomes, includingvectors and selection sequences or markers operable for stableintegration into a host chromosome. Additionally, the expression vectorscan include one or more selectable marker genes and appropriateexpression control sequences. Selectable marker genes also can beincluded that, for example, provide resistance to antibiotics or toxins,complement auxotrophic deficiencies, or supply critical nutrients not inthe culture media. Expression control sequences can include constitutiveand inducible promoters, transcription enhancers, transcriptionterminators, and the like which are well known in the art. When two ormore exogenous encoding nucleic acids are to be co-expressed, bothnucleic acids can be inserted, for example, into a single expressionvector or in separate expression vectors. For single vector expression,the encoding nucleic acids can be operationally linked to one commonexpression control sequence or linked to different expression controlsequences, such as one inducible promoter and one constitutive promoter.The transformation of exogenous nucleic acid sequences involved in ametabolic or synthetic pathway can be confirmed using methods well knownin the art. Such methods include, for example, nucleic acid analysissuch as Northern blots or polymerase chain reaction (PCR) amplificationof mRNA, or immunoblotting for expression of gene products, or othersuitable analytical methods to test the expression of an introducednucleic acid sequence or its corresponding gene product. It isunderstood by those skilled in the art that the exogenous nucleic acidis expressed in a sufficient amount to produce the desired product, andit is further understood that expression levels can be optimized toobtain sufficient expression using methods well known in the art and asdisclosed herein.

In some embodiments, the present invention provides a method forenhancing carbon flux through acetyl-CoA that includes culturing theaforementioned non-naturally occurring microbial organisms underconditions and for a sufficient period of time to produce a producthaving acetyl-CoA as a building block. Such culturing can be in asubstantially anaerobic culture medium and can include organisms havingany number of exogenous nucleic acids as described herein above.

As described above, these cultured organisms can have an isopropanolpathway, a 1,3-butanediol pathway; a 1,4-butanediol pathway, a4-hydroxybutrate pathway, or any other functional pathway that utilizesacetyl-CoA. The culturing of these microbial organism can be performedwith a carbon feedstock selected from CO, CO₂, and H₂, synthesis gascomprising CO and H₂, and synthesis gas comprising CO, CO₂, and H₂.

Suitable purification and/or assays to test for the production of1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanol can beperformed using well known methods. Suitable replicates such astriplicate cultures can be grown for each engineered strain to betested. For example, product and byproduct formation in the engineeredproduction host can be monitored. The final product and intermediates,and other organic compounds, can be analyzed by methods such as HPLC(High Performance Liquid Chromatography), GC-MS (Gas Chromatography-MassSpectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) orother suitable analytical methods using routine procedures well known inthe art. The release of product in the fermentation broth can also betested with the culture supernatant. Byproducts and residual glucose canbe quantified by HPLC using, for example, a refractive index detectorfor glucose and alcohols, and a UV detector for organic acids (Lin etal., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay anddetection methods well known in the art. The individual enzyme orprotein activities from the exogenous DNA sequences can also be assayedusing methods well known in the art.

The 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolcan be separated from other components in the culture using a variety ofmethods well known in the art. Such separation methods include, forexample, extraction procedures as well as methods that includecontinuous liquid-liquid extraction, pervaporation, membrane filtration,membrane separation, reverse osmosis, electrodialysis, distillation,crystallization, centrifugation, extractive filtration, ion exchangechromatography, size exclusion chromatography, adsorptionchromatography, and ultrafiltration. All of the above methods are wellknown in the art.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol producers can be cultured for thebiosynthetic production of 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol.

For the production of 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol,or isopropanol, the recombinant strains are cultured in a medium withcarbon source and other essential nutrients. It is sometimes desirableand can be highly desirable to maintain anaerobic conditions in thefermenter to reduce the cost of the overall process. Such conditions canbe obtained, for example, by first sparging the medium with nitrogen andthen sealing the flasks with a septum and crimp-cap. For strains wheregrowth is not observed anaerobically, microaerobic or substantiallyanaerobic conditions can be applied by perforating the septum with asmall hole for limited aeration. Exemplary anaerobic conditions havebeen described previously and are well-known in the art. Exemplaryaerobic and anaerobic conditions are described, for example, in UnitedState publication 2009/0047719, filed Aug. 10, 2007. Fermentations canbe performed in a batch, fed-batch or continuous manner, as disclosedherein.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

The growth medium can include, for example, any carbohydrate sourcewhich can supply a source of carbon to the non-naturally occurringmicroorganism. Such sources include, for example, sugars such asglucose, xylose, arabinose, galactose, mannose, fructose, sucrose andstarch. Other sources of carbohydrate include, for example, renewablefeedstocks and biomass. Exemplary types of biomasses that can be used asfeedstocks in the methods of the invention include cellulosic biomass,hemicellulosic biomass and lignin feedstocks or portions of feedstocks.Such biomass feedstocks contain, for example, carbohydrate substratesuseful as carbon sources such as glucose, xylose, arabinose, galactose,mannose, fructose and starch. Given the teachings and guidance providedherein, those skilled in the art will understand that renewablefeedstocks and biomass other than those exemplified above also can beused for culturing the microbial organisms of the invention for theproduction of 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol.

In addition to renewable feedstocks such as those exemplified above, the1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolmicrobial organisms of the invention also can be modified for growth onsyngas as its source of carbon. In this specific embodiment, one or moreproteins or enzymes are expressed in the 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol producing organisms toprovide a metabolic pathway for utilization of syngas or other gaseouscarbon source.

Given the teachings and guidance provided herein, those skilled in theart will understand that a non-naturally occurring microbial organismcan be produced that secretes the biosynthesized compounds of theinvention when grown on a carbon source such as a carbohydrate. Suchcompounds include, for example, 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol and any of the intermediate metabolitesin the 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolpathway. All that is required is to engineer in one or more of therequired enzyme or protein activities to achieve biosynthesis of thedesired compound or intermediate including, for example, inclusion ofsome or all of the 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol biosynthetic pathways. Accordingly, the invention provides anon-naturally occurring microbial organism that produces and/or secretes1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanol whengrown on a carbohydrate or other carbon source and produces and/orsecretes any of the intermediate metabolites shown in the1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolpathway when grown on a carbohydrate or other carbon source. The1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolproducing microbial organisms of the invention can initiate synthesisfrom an intermediate.

The non-naturally occurring microbial organisms of the invention areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding a 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol pathway enzyme orprotein in sufficient amounts to produce 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol. It is understood thatthe microbial organisms of the invention are cultured under conditionssufficient to produce 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol,or isopropanol. Following the teachings and guidance provided herein,the non-naturally occurring microbial organisms of the invention canachieve biosynthesis of 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol resulting in intracellular concentrationsbetween about 0.1-200 mM or more. Generally, the intracellularconcentration of 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol is between about 3-150 mM, particularly between about 5-125mM and more particularly between about 8-100 mM, including about 10 mM,20 mM, 50 mM, 80 mM, or more. Intracellular concentrations between andabove each of these exemplary ranges also can be achieved from thenon-naturally occurring microbial organisms of the invention.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. publication2009/0047719, filed Aug. 10, 2007. Any of these conditions can beemployed with the non-naturally occurring microbial organisms as well asother anaerobic conditions well known in the art. Under such anaerobicor substantially anaerobic conditions, the 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol producers cansynthesize 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol at intracellular concentrations of 5-10 mM or more as wellas all other concentrations exemplified herein. It is understood that,even though the above description refers to intracellularconcentrations, 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol producing microbial organisms can produce 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol intracellularly and/orsecrete the product into the culture medium.

In addition to the culturing and fermentation conditions disclosedherein, growth condition for achieving biosynthesis of 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol can include theaddition of an osmoprotectant to the culturing conditions. In certainembodiments, the non-naturally occurring microbial organisms of theinvention can be sustained, cultured or fermented as described herein inthe presence of an osmoprotectant. Briefly, an osmoprotectant refers toa compound that acts as an osmolyte and helps a microbial organism asdescribed herein survive osmotic stress. Osmoprotectants include, butare not limited to, betaines, amino acids, and the sugar trehalose.Non-limiting examples of such are glycine betaine, praline betaine,dimethylthetin, dimethyl slfonioproprionate,3-dimethylsulfonio-2-methylproprionate, pipecolic acid,dimethylsulfonioacetate, choline, L-carnitine and ectoine. In oneaspect, the osmoprotectant is glycine betaine. It is understood to oneof ordinary skill in the art that the amount and type of osmoprotectantsuitable for protecting a microbial organism described herein fromosmotic stress will depend on the microbial organism used. The amount ofosmoprotectant in the culturing conditions can be, for example, no morethan about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM,no more than about 1.5 mM, no more than about 2.0 mM, no more than about2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no morethan about 7.0 mM, no more than about 10 mM, no more than about 50 mM,no more than about 100 mM or no more than about 500 mM.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol includes anaerobic culture or fermentation conditions. Incertain embodiments, the non-naturally occurring microbial organisms ofthe invention can be sustained, cultured or fermented under anaerobic orsubstantially anaerobic conditions. Briefly, anaerobic conditions referto an environment devoid of oxygen. Substantially anaerobic conditionsinclude, for example, a culture, batch fermentation or continuousfermentation such that the dissolved oxygen concentration in the mediumremains between 0 and 10% of saturation. Substantially anaerobicconditions also includes growing or resting cells in liquid medium or onsolid agar inside a sealed chamber maintained with an atmosphere of lessthan 1% oxygen. The percent of oxygen can be maintained by, for example,sparging the culture with an N₂/CO₂ mixture or other suitable non-oxygengas or gases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol. Exemplary growth procedures include, forexample, fed-batch fermentation and batch separation; fed-batchfermentation and continuous separation, or continuous fermentation andcontinuous separation. All of these processes are well known in the art.Fermentation procedures are particularly useful for the biosyntheticproduction of commercial quantities of 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol. Generally, and aswith non-continuous culture procedures, the continuous and/ornear-continuous production of 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol will include culturing a non-naturallyoccurring 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol producing organism of the invention in sufficient nutrientsand medium to sustain and/or nearly sustain growth in an exponentialphase. Continuous culture under such conditions can be included, forexample, growth for 1 day, 2, 3, 4, 5, 6 or 7 days or more.Additionally, continuous culture can include longer time periods of 1week, 2, 3, 4 or 5 or more weeks and up to several months.Alternatively, organisms of the invention can be cultured for hours, ifsuitable for a particular application. It is to be understood that thecontinuous and/or near-continuous culture conditions also can includeall time intervals in between these exemplary periods. It is furtherunderstood that the time of culturing the microbial organism of theinvention is for a sufficient period of time to produce a sufficientamount of product for a desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol can be utilized in, for example,fed-batch fermentation and batch separation; fed-batch fermentation andcontinuous separation, or continuous fermentation and continuousseparation. Examples of batch and continuous fermentation procedures arewell known in the art.

In addition to the above fermentation procedures using the1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolproducers of the invention for continuous production of substantialquantities of 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol, the 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol producers also can be, for example, simultaneously subjectedto chemical synthesis procedures to convert the product to othercompounds or the product can be separated from the fermentation cultureand sequentially subjected to chemical conversion to convert the productto other compounds, if desired.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of 1,4-butanediol,4-hydroxybutyrate, 1,3-butanediol, or isopropanol.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion or disruption strategies that result ingenetically stable microorganisms which overproduce the target product.Specifically, the framework examines the complete metabolic and/orbiochemical network of a microorganism in order to suggest geneticmanipulations that force the desired biochemical to become an obligatorybyproduct of cell growth. By coupling biochemical production with cellgrowth through strategically placed gene deletions or other functionalgene disruption, the growth selection pressures imposed on theengineered strains after long periods of time in a bioreactor lead toimprovements in performance as a result of the compulsory growth-coupledbiochemical production. Lastly, when gene deletions are constructedthere is a negligible possibility of the designed strains reverting totheir wild-type states because the genes selected by OptKnock are to becompletely removed from the genome. Therefore, this computationalmethodology can be used to either identify alternative pathways thatlead to biosynthesis of a desired product or used in connection with thenon-naturally occurring microbial organisms for further optimization ofbiosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat allow an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. publication 2002/0168654, filed Jan. 10, 2002, in InternationalPatent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. publication2009/0047719, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

As disclosed herein, a nucleic acid encoding a desired activity of a1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolpathway can be introduced into a host organism. In some cases, it can bedesirable to modify an activity of a 1,4-butanediol, 4-hydroxybutyrate,1,3-butanediol, or isopropanol pathway enzyme or protein to increaseproduction of 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, orisopropanol. For example, known mutations that increase the activity ofa protein or enzyme can be introduced into an encoding nucleic acidmolecule. Additionally, optimization methods can be applied to increasethe activity of an enzyme or protein and/or decrease an inhibitoryactivity, for example, decrease the activity of a negative regulator.

One such optimization method is directed evolution. Directed evolutionis a powerful approach that involves the introduction of mutationstargeted to a specific gene in order to improve and/or alter theproperties of an enzyme. Improved and/or altered enzymes can beidentified through the development and implementation of sensitivehigh-throughput screening assays that allow the automated screening ofmany enzyme variants (for example, >10⁴). Iterative rounds ofmutagenesis and screening typically are performed to afford an enzymewith optimized properties. Computational algorithms that can help toidentify areas of the gene for mutagenesis also have been developed andcan significantly reduce the number of enzyme variants that need to begenerated and screened. Numerous directed evolution technologies havebeen developed (for reviews, see Hibbert et al., Biomol. Eng. 22:11-19(2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical andbiotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press;Often and Quax. Biomol. Eng. 22:1-9 (2005).; and Sen et al., ApplBiochem. Biotechnol 143:212-223 (2007)) to be effective at creatingdiverse variant libraries, and these methods have been successfullyapplied to the improvement of a wide range of properties across manyenzyme classes. Enzyme characteristics that have been improved and/oraltered by directed evolution technologies include, for example:selectivity/specificity, for conversion of non-natural substrates;temperature stability, for robust high temperature processing; pHstability, for bioprocessing under lower or higher pH conditions;substrate or product tolerance, so that high product titers can beachieved; binding (K_(m)), including broadening substrate binding toinclude non-natural substrates; inhibition (KO, to remove inhibition byproducts, substrates, or key intermediates; activity (k_(cat)), toincreases enzymatic reaction rates to achieve desired flux; expressionlevels, to increase protein yields and overall pathway flux; oxygenstability, for operation of air sensitive enzymes under aerobicconditions; and anaerobic activity, for operation of an aerobic enzymein the absence of oxygen.

A number of exemplary methods have been developed for the mutagenesisand diversification of genes to target desired properties of specificenzymes. Such methods are well known to those skilled in the art. Any ofthese can be used to alter and/or optimize the activity of a1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, or isopropanolpathway enzyme or protein. Such methods include, but are not limited toEpPCR, which introduces random point mutations by reducing the fidelityof DNA polymerase in PCR reactions (Pritchard et al., J. Theor. Biol.234:497-509 (2005)); Error-prone Rolling Circle Amplification (epRCA),which is similar to epPCR except a whole circular plasmid is used as thetemplate and random 6-mers with exonuclease resistant thiophosphatelinkages on the last 2 nucleotides are used to amplify the plasmidfollowed by transformation into cells in which the plasmid isre-circularized at tandem repeats (Fujii et al., Nucleic Acids Res.32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497 (2006)); DNAor Family Shuffling, which typically involves digestion of two or morevariant genes with nucleases such as Dnase I or EndoV to generate a poolof random fragments that are reassembled by cycles of annealing andextension in the presence of DNA polymerase to create a library ofchimeric genes (Stemmer, Proc. Natl. Acad. Sci. U.S.A. 91:10747-10751(1994); and Stemmer, Nature 370:389-391 (1994)); Staggered Extension(StEP), which entails template priming followed by repeated cycles of 2step PCR with denaturation and very short duration ofannealing/extension (as short as 5 sec) (Zhao et al., Nat. Biotechnol.16:258-261 (1998)); Random Priming Recombination (RPR), in which randomsequence primers are used to generate many short DNA fragmentscomplementary to different segments of the template (Shao et al.,Nucleic Acids Res. 26:681-683 (1998)).

Additional methods include Heteroduplex Recombination, in whichlinearized plasmid DNA is used to form heteroduplexes that are repairedby mismatch repair (Volkov et al, Nucleic Acids Res. 27:e18 (1999); andVolkov et al., Methods Enzymol. 328:456-463 (2000)); RandomChimeragenesis on Transient Templates (RACHITT), which employs Dnase Ifragmentation and size fractionation of single stranded DNA (ssDNA)(Coco et al., Nat. Biotechnol. 19:354-359 (2001)); Recombined Extensionon Truncated templates (RETT), which entails template switching ofunidirectionally growing strands from primers in the presence ofunidirectional ssDNA fragments used as a pool of templates (Lee et al.,J. Molec. Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide GeneShuffling (DOGS), in which degenerate primers are used to controlrecombination between molecules; (Bergquist and Gibbs, Methods Mol.Biol. 352:191-204 (2007); Bergquist et al., Biomol. Eng. 22:63-72(2005); Gibbs et al., Gene 271:13-20 (2001)); Incremental Truncation forthe Creation of Hybrid Enzymes (ITCHY), which creates a combinatoriallibrary with 1 base pair deletions of a gene or gene fragment ofinterest (Ostermeier et al., Proc. Natl. Acad. Sci. U.S.A. 96:3562-3567(1999); and Ostermeier et al., Nat. Biotechnol. 17:1205-1209 (1999));Thio-Incremental Truncation for the Creation of Hybrid Enzymes(THIO-ITCHY), which is similar to ITCHY except that phosphothioate dNTPsare used to generate truncations (Lutz et al., Nucleic Acids Res. 29:E16(2001)); SCRATCHY, which combines two methods for recombining genes,ITCHY and DNA shuffling (Lutz et al., Proc. Natl. Acad. Sci. U.S.A.98:11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in whichmutations made via epPCR are followed by screening/selection for thoseretaining usable activity (Bergquist et al., Biomol. Eng. 22:63-72(2005)); Sequence Saturation Mutagenesis (SeSaM), a random mutagenesismethod that generates a pool of random length fragments using randomincorporation of a phosphothioate nucleotide and cleavage, which is usedas a template to extend in the presence of “universal” bases such asinosine, and replication of an inosine-containing complement givesrandom base incorporation and, consequently, mutagenesis (Wong et al.,Biotechnol. J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26(2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)); SyntheticShuffling, which uses overlapping oligonucleotides designed to encode“all genetic diversity in targets” and allows a very high diversity forthe shuffled progeny (Ness et al., Nat. Biotechnol. 20:1251-1255(2002)); Nucleotide Exchange and Excision Technology NexT, whichexploits a combination of dUTP incorporation followed by treatment withuracil DNA glycosylase and then piperidine to perform endpoint DNAfragmentation (Muller et al., Nucleic Acids Res. 33:e117 (2005)).

Further methods include Sequence Homology-Independent ProteinRecombination (SHIPREC), in which a linker is used to facilitate fusionbetween two distantly related or unrelated genes, and a range ofchimeras is generated between the two genes, resulting in libraries ofsingle-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460(2001)); Gene Site Saturation Mutagenesis™ (GSSM™), in which thestarting materials include a supercoiled double stranded DNA (dsDNA)plasmid containing an insert and two primers which are degenerate at thedesired site of mutations (Kretz et al., Methods Enzymol. 388:3-11(2004)); Combinatorial Cassette Mutagenesis (CCM), which involves theuse of short oligonucleotide cassettes to replace limited regions with alarge number of possible amino acid sequence alterations (Reidhaar-Olsonet al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al.Science 241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis(CMCM), which is essentially similar to CCM and uses epPCR at highmutation rate to identify hot spots and hot regions and then extensionby CMCM to cover a defined region of protein sequence space (Reetz etal., Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); the MutatorStrains technique, in which conditional is mutator plasmids, utilizingthe mutD5 gene, which encodes a mutant subunit of DNA polymerase III, toallow increases of 20 to 4000-× in random and natural mutation frequencyduring selection and block accumulation of deleterious mutations whenselection is not required (Selifonova et al., Appl. Environ. Microbiol.67:3645-3649 (2001)); Low et al., J. Mol. Biol. 260:359-3680 (1996)).

Additional exemplary methods include Look-Through Mutagenesis (LTM),which is a multidimensional mutagenesis method that assesses andoptimizes combinatorial mutations of selected amino acids (Rajpal etal., Proc. Natl. Acad. Sci. U.S.A. 102:8466-8471 (2005)); GeneReassembly, which is a DNA shuffling method that can be applied tomultiple genes at one time or to create a large library of chimeras(multiple mutations) of a single gene (Tunable GeneReassembly™ (TGR™)Technology supplied by Verenium Corporation), in Silico Protein DesignAutomation (PDA), which is an optimization algorithm that anchors thestructurally defined protein backbone possessing a particular fold, andsearches sequence space for amino acid substitutions that can stabilizethe fold and overall protein energetics, and generally works mosteffectively on proteins with known three-dimensional structures (Hayeset al., Proc. Natl. Acad. Sci. U.S.A. 99:15926-15931 (2002)); andIterative Saturation Mutagenesis (ISM), which involves using knowledgeof structure/function to choose a likely site for enzyme improvement,performing saturation mutagenesis at chosen site using a mutagenesismethod such as Stratagene QuikChange (Stratagene; San Diego Calif.),screening/selecting for desired properties, and, using improvedclone(s), starting over at another site and continue repeating until adesired activity is achieved (Reetz et al., Nat. Protoc. 2:891-903(2007); and Reetz et al., Angew. Chem. Int. Ed Engl. 45:7745-7751(2006)).

Any of the aforementioned methods for mutagenesis can be used alone orin any combination. Additionally, any one or combination of the directedevolution methods can be used in conjunction with adaptive evolutiontechniques, as described herein.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties, includingGenBank and GI number publications, are hereby incorporated by referencein this application in order to more fully describe the state of the artto which this invention pertains. Although the invention has beendescribed with reference to the examples provided above, it should beunderstood that various modifications can be made without departing fromthe spirit of the invention.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLES

Non-naturally occurring organism to produce 1,3-butanediol, isopropanol,4-hydroxybutyrate or 1,4-butanediol from acetyl-CoA

In the following Examples, pathways for formation of 1,3-BDO (FIG. 1A),isopropanol (FIG. 1B), 4-HB (FIG. 1C), and 1,4-BDO (FIG. 1D) aredescribed from the intermediate, acetyl-CoA. The maximum theoreticalyield of each of these product molecules from glucose is 1 mole per moleusing only the metabolic pathways proceeding from acetyl-CoA asdescribed herein. Specifically, 2 moles of acetyl-CoA are derived permole of glucose via glycolysis and 2 moles of acetyl-CoA are used permole of 1,3-butanediol, isopropanol, 4-hydroxybutyrate, or1,4-butanediol. The net conversions are described by the followingstoichiometric equations:

C₆H₁₂O₆→C₄H₁₀O₂+CH₂O₂+CO₂  1,3-Butanediol:

C₆H₁₂O₆+1.5O₂→C₃H₈O+3CO₂+2H₂O  Isopropanol:

C₆H₁₂O₆+1.5O₂→C₄H₈O₃+2CO₂+2H₂O  4-Hydroxybutyate:

C₆H₁₂O₆→C₄H₁₀O₂+CH₂O₂+CO₂  1,4-Butanediol:

Example I 1,3-Butanediol Synthesis Pathway

1,3-butanediol production can be achieved in recombinant E. coli byalternative pathways as described in FIG. 1A. All pathways first converttwo molecules of acetyl-CoA into one molecule of acetoacetyl-CoAemploying a thiolase.

Acetoacetyl-CoA thiolase converts two molecules of acetyl-CoA into onemolecule each of acetoacetyl-CoA and CoA. Exemplary acetoacetyl-CoAthiolase enzymes include the gene products of atoB from E. coli (Martinet al., Nat. Biotechnol. 21:796-802 (2003), thlA and thlB from C.acetobutylicum (Hanai et al., Appl. Environ. Microbiol. 73:7814-7818(2007); Winzer et al., J. Mol. Microbiol. Biotechnol. 2:531-541 (2000),and ERG10 from S. cerevisiae (Hiser et al., J. Biol. Chem.269:31383-31389 (1994).

Protein GenBank ID GI number Organism AtoB NP_416728 16130161Escherichia coli ThlA NP_349476.1 15896127 Clostridium acetobutylicumThlB NP_149242.1 15004782 Clostridium acetobutylicum ERG10 NP_015297 6325229 Saccharomyces cerevisiae

Acetoacetyl-CoA can first be reduced to 3-hydroxybutyryl-CoA byacetoacetyl-CoA reductase (ketone reducing). This can subsequently beconverted to 3-hydroxybutyraldehyde via a CoA-dependent aldehydereductase called 3-hydroxybutyryl-CoA reductase. 3-hydroxybutyraldehydecan eventually be reduced to the product 1,3-BDO by3-hydroxybutyraldehyde reductase. Alternatively, 3-hydroxybutyryl-CoAcan be reduced directly to 1,3-BDO by an alcohol-forming CoA-dependent3-hydroxybutyryl-CoA reductase. The gene candidates for each of thesteps in the pathway are described below.

Acetoacetyl-CoA reductase catalyzing the reduction of acetoacetyl-CoA to3-hydroxybutyryl-CoA participates in the acetyl-CoA fermentation pathwayto butyrate in several species of Clostridia and has been studied indetail (Jones and Woods, Microbiol. Rev. 50:484-524 (1986)). The enzymefrom Clostridium acetobutylicum, encoded by hbd, has been cloned andfunctionally expressed in E. coli (Youngleson et al., J. Bacteriol.171:6800-6807 (1989)). Additionally, subunits of two fatty acidoxidation complexes in E. coli, encoded by fadB and fads, function as3-hydroxyacyl-CoA dehydrogenases (Binstockand Schulz, Methods Enzymol.71 Pt C:403-411 (1981)). Yet other gene candidates demonstrated toreduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA are phbB from Zoogloearamigera (Ploux et al., Eur. J. Biochem. 174:177-182 (1988) and phaBfrom Rhodobacter sphaeroides (Alber et al., Mol. Microbiol. 61:297-309(2006). The former gene candidate is NADPH-dependent, its nucleotidesequence has been determined (Peoples and Sinskey, Mol. Microbiol.3:349-357 (1989) and the gene has been expressed in E. coli. Substratespecificity studies on the gene led to the conclusion that it couldaccept 3-oxopropionyl-CoA as a substrate besides acetoacetyl-CoA (Plouxet al., Eur. J. Biochem. 174:177-182 (1988)). Additional gene candidatesinclude Hbd1 (C-terminal domain) and Hbd2 (N-terminal domain) inClostridium kluyveri (Hillmer and Gottschalk, Biochim. Biophys. Acta3334:12-23 (1974)) and HSD17B10 in Bos taurus (Wakil et al., J. Biol.Chem. 207:631-638 (1954)).

Protein Genbank ID GI number Organism fadB P21177.2   119811 Escherichiacoli fadJ P77399.1  3334437 Escherichia coli Hbd2 EDK34807.1 146348271Clostridium kluyveri Hbd1 EDK32512.1 146345976 Clostridium kluyveri hbdP52041.2 Clostridium acetobutylicum HSD17B10 O02691.3  3183024 BosTaurus phbB P23238.1   130017 Zoogloea ramigera phaB YP_353825.1 77464321 Rhodobacter sphaeroides

A number of similar enzymes have been found in other species ofClostridia and in Metallosphaera sedula (Berg et al., Science318:1782-1786 (2007).

Protein GenBank ID GI number Organism hbd NP_349314.1 NP_349314.1Clostridium acetobutylicum hbd AAM14586.1 AAM14586.1 Clostridiumbeijerinckii Msed_1423 YP_001191505 YP_001191505 Metallosphaera sedulaMsed_0399 YP_001190500 YP_001190500 Metallosphaera sedula Msed_0389YP_001190490 YP_001190490 Metallosphaera sedula Msed_1993 YP_001192057YP_001192057 Metallosphaera sedula

Several acyl-CoA dehydrogenases are capable of reducing an acyl-CoA toits corresponding aldehyde. Exemplary genes that encode such enzymesinclude the Acinetobacter calcoaceticus acr1 encoding a fatty acyl-CoAreductase (Reiser and Somerville, J. Bacteriol. 179:2969-2975 (1997),the Acinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al., Appl.Environ. Microbiol. 68:1192-1195 (2002), and a CoA- and NADP-dependentsuccinate semialdehyde dehydrogenase encoded by the sucD gene inClostridium kluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-880(1996); Sohling and Gottschalk, J. Bacteriol. 1778:871-880 (1996)). SucDof P. gingivalis is another succinate semialdehyde dehydrogenase(Takahashi et al., J. Bacteriol. 182:4704-4710 (2000). The enzymeacylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG,is yet another candidate as it has been demonstrated to oxidize andacylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehydeand formaldehyde (Powlowski et al., J. Bacteriol. 175:377-385 (1993)).In addition to reducing acetyl-CoA to ethanol, the enzyme encoded byadhE in Leuconostoc mesenteroides has been shown to oxidize the branchedchain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J.Gen. Appl. Microbiol. 18:45-55 (1972); Koo et al., Biotechnol. Lett.27:505-510 (2005)). Butyraldehyde dehydrogenase catalyzes a similarreaction, conversion of butyryl-CoA to butyraldehyde, in solventogenicorganisms such as Clostridium saccharoperbutylacetonicum (Kosaka et al.,Biosci. Biotechnol. Biochem. 71:58-68 (2007)).

Protein GenBank ID GI number Organism acr1 YP_047869.1  50086359Acinetobacter calcoaceticus acr1 AAC45217  1684886 Acinetobacter baylyiacr1 BAB85476.1  18857901 Acinetobacter sp. Strain M-1 sucD P38947.1172046062 Clostridium kluyveri sucD NP_904963.1  34540484 Porphyromonasgingivalis bphG BAA03892.1   425213 Pseudomonas sp adhE AAV66076.1 55818563 Leuconostoc mesenteroides bld AAP42563.1  31075383 Clostridiumsaccharoperbutylacetonicum

An additional enzyme type that converts an acyl-CoA to its correspondingaldehyde is malonyl-CoA reductase which transforms malonyl-CoA tomalonic semialdehyde. Malonyl-CoA reductase is a key enzyme inautotrophic carbon fixation via the 3-hydroxypropionate cycle inthermoacidophilic archaeal bacteria (Berg et al., Science 318:1782-1786(2007); Thauer, Science 318:1732-1733 (2007)). The enzyme utilizes NADPHas a cofactor and has been characterized in Metallosphaera andSulfolobus spp (Alber et al., J. Bacteriol. 188:8551-8559 (2006); Hugleret al., J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded byMsed 0709 in Metallosphaera sedula (Alber et al., supra (2006); Berg etal., Science 318:1782-1786 (2007)). A gene encoding a malonyl-CoAreductase from Sulfolobus tokodaii was cloned and heterologouslyexpressed in E. coli (Alber et al., J. Bacteriol. 188:8551-8559 (2006)).This enzyme has also been shown to catalyze the conversion ofmethylmalonyl-CoA to its corresponding aldehyde (WO 2007/141208 (2007)).Although the aldehyde dehydrogenase functionality of these enzymes issimilar to the bifunctional dehydrogenase from Chloroflexus aurantiacus,there is little sequence similarity. Both malonyl-CoA reductase enzymecandidates have high sequence similarity to aspartate-semialdehydedehydrogenase, an enzyme catalyzing the reduction and concurrentdephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde.Additional gene candidates can be found by sequence homology to proteinsin other organisms including Sulfolobus solfataricus and Sulfolobusacidocaldarius and have been listed below. Yet another candidate forCoA-acylating aldehyde dehydrogenase is the ald gene from Clostridiumbeijerinckii (Toth et al., Appl. Environ. Microbiol. 65:4973-4980(1999). This enzyme has been reported to reduce acetyl-CoA andbutyryl-CoA to their corresponding aldehydes. This gene is very similarto eutE that encodes acetaldehyde dehydrogenase of Salmonellatyphimurium and E. coli (Toth et al., supra).

Protein GenBank ID GI number Organism Msed_0709 YP_001190808.1 146303492Metallosphaera sedula mcr NP_378167.1  15922498 Sulfolobus tokodaiiasd-2 NP_343563.1  15898958 Sulfolobus solfataricus Saci_2370YP_256941.1  70608071 Sulfolobus acidocaldarius Ald AAT66436  9473535Clostridium beijerinckii eutE AAA80209   687645 Salmonella typhimuriumeutE P77445  2498347 Escherichia coli

Enzymes exhibiting 3-hydroxybutyraldehyde reductase activity (EC1.1.1.61) have been characterized in Ralstonia eutropha (Bravo et al.,J. Forensic Sci. 49:379-387 (2004)), Clostridium kluyveri (Wolff andKenealy, Protein Expr. Purif. 6:206-212 (1995)) and Arabidopsis thaliana(Breitkreuz et al., J. Biol. Chem. 278:41552-41556 (2003). Yet anothergene candidate is the alcohol dehydrogenase adhI from Geobacillusthermoglucosidasius (Jeon et al., J. Biotechnol. 135:127-133 (2008).

Protein GenBank ID GI number Organism 4hbd YP_726053.1 113867564Ralstonia eutropha H16 4hbd L21902.1 146348486 Clostridium kluyveri DSM555 4hbd Q94B07  75249805 Arabidopsis thaliana adhI AAR91477.1  40795502Geobacillus thermoglucosidasius M10EXG

Another exemplary enzyme is 3-hydroxyisobutyrate dehydrogenase whichcatalyzes the reversible oxidation of 3-hydroxyisobutyrate tomethylmalonate semialdehyde. This enzyme participates in valine, leucineand isoleucine degradation and has been identified in bacteria,eukaryotes, and mammals. The enzyme encoded by P84067 from Thermusthermophilus HB8 has been structurally characterized (Lokanath et al.,J. Mol. Biol. 352:905-917 (2005)). The reversibility of the human3-hydroxyisobutyrate dehydrogenase was demonstrated usingisotopically-labeled substrate (Manning and Pollitt, Biochem. J.231:481-484 (1985)). Additional genes encoding this enzyme include 3hidhin Homo sapiens (Hawes et al., Methods Enzymol. 324:218-228 (2000) andOryctolagus cuniculus (Hawes et al., supra; Chowdhury et al., Biosci.Biotechnol. 60:2043-2047 (1996), mmsb in Pseudomonas aeruginosa, anddhat in Pseudomonas putida (Aberhart and Hsu, I Chem. Soc. [Perkin 1]6:1404-1406 (1979); Chowdhury et al., supra; Chowdhury et al., Biosci.Biotechnol. Biochem. 67:438-441 (2003)).

Protein GenBank ID GI number Organism P84067 P84067 75345323 Thermusthermophilus mmsb P28811.1  127211 Pseudomonas aeruginosa dhat Q59477.1 2842618 Pseudomonas putida 3hidh P31937.2 12643395 Homo sapiens 3hidhP32185.1  416872 Oryctolagus cuniculus

Other exemplary genes encoding enzymes that catalyze the conversion ofan aldehyde to alcohol (i.e., alcohol dehydrogenase or equivalentlyaldehyde reductase) include alrA encoding a medium-chain alcoholdehydrogenase for C2-C14 (Tani et al., App. Environ. Microbiol.66:5231-5235 (2000), ADH2 from Saccharomyces cerevisiae (Atsumi et al.,Nature 451:86-89 (2008)), yqhD from E. coli which has preference formolecules longer than C3 (Sulzenbacher et al., J. Mol. Biol. 342:489-502(2004), and bdh I and bdh II from C. acetobutylicum which convertsbutyraldehyde into butanol (Walter et al., J. Bacteriol. 174:7149-7158(1992)). The gene product of yqhD catalyzes the reduction ofacetaldehyde, malondialdehyde, propionaldehyde, butyraldehyde, andacrolein using NADPH as the cofactor (Perez et al., J. Biol. Chem.283:7346-7353 (2008)). ADH1 from Zymomonas mobilis has been demonstratedto have activity on a number of aldehydes including formaldehyde,acetaldehyde, propionaldehyde, butyraldehyde, and acrolein (Kinoshita etal., Appl. Microbiol. Biotechnol. 22:249-254 (1985)). The proteinsequences for each of these exemplary gene products, if available, canbe found using the following GenBank accession numbers:

Protein GenBank ID GI number Organism alrA BAB12273.1 9967138Acinetobacter sp. strain M-1 ADH2 NP_014032.1 6323961 Saccharomycescerevisiae yqhD NP_417484.1 16130909 Escherichia coli bdh I NP_349892.115896543 Clostridium acetobutylicum bdh II NP_349891.1 15896542Clostridium acetobutylicum adhA YP_162971.1 56552132 Zymomonas mobilis

Exemplary two-step oxidoreductases that convert an acyl-CoA to alcoholinclude those that transform substrates such as acetyl-CoA to ethanol(e.g., adhE from E. coli (Kessler et al., FEBS Lett. 281:59-63 (1991))and butyryl-CoA to butanol (e.g. adhE2 from C. acetobutylicum (Fontaineet al., J. Bacteriol. 184:821-830 (2002)). In addition to reducingacetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostocmesenteroides has been shown to oxidize the branched chain compoundisobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J. Gen. Appl.Microbiol. 18:43-55 (1972); Koo et al., Biotechnol. Lett. 27:505-510(2005)).

Protein GenBank ID GI number Organism adhE NP_415757.1 16129202Escherichia coli adhE2 AAK09379.1 12958626 Clostridium acetobutylicumadhE AAV66076.1 55818563 Leuconostoc mesenteroides

Another exemplary enzyme can convert malonyl-CoA to 3-HP. AnNADPH-dependent enzyme with this activity has characterized inChloroflexus aurantiacus where it participates in the3-hydroxypropionate cycle (Hugler et al., J. Bacteriol. 184:2404-2410(2002); Strauss and Fuchs, Eur. J. Biochem. 215:633-643 (1993). Thisenzyme, with a mass of 300 kDa, is highly substrate-specific and showslittle sequence similarity to other known oxidoreductases (Hugler, supra(2002)). No enzymes in other organisms have been shown to catalyze thisspecific reaction; however there is bioinformatic evidence that otherorganisms may have similar pathways (Klatt et al., Environ. Microbiol.9:2067-2078 (2007)). Enzyme candidates in other organisms includingRoseiflexus castenholzii, Erythrobacter sp. NAP1 and marine gammaproteobacterium HTCC2080 can be inferred by sequence similarity.

Protein GenBank ID GI number Organism Rcas_2929 YP_001433009.1 156742880Roseiflexus castenholzii NAP1_02720 ZP_01039179.1 85708113 Erythrobactersp. NAP1 MGP2080_00535 ZP_01626393.1 119504313 marine gammaproteobacterium HTCC2080

Longer chain acyl-CoA molecules can be reduced by enzymes such as thejojoba (Simmondsia chinensis) FAR which encodes an alcohol-forming fattyacyl-CoA reductase. Its overexpression in E. coli resulted in FARactivity and the accumulation of fatty alcohol (Metz et al., PlantPhysiol. 122:635-644 (2000)).

FAR AAD38039.1 5020215 Simmondsia chinensis

The reduction of acetoacetyl-CoA into 3-oxobutyraldehyde can beaccomplished via the CoA-dependent aldehyde forming acetoacetyl-CoAreductase. 3-oxobutyraldehyde is next reduced to 3-hydroxybutyraldehydeby 3-oxobutyraldehyde reductase (ketone reducing), and eventually, thisintermediate is reduced to 1,3-butanediol by a 3-hydroxybutyraldehydereductase. The candidates for each of these steps are listed below.

The enzymes for acetoacetyl-CoA reductase (CoA-dependent, aldehydeforming) are the same as that for the aldehyde forming3-hydroxybutyryl-CoA reductase described above.

There exist several exemplary alcohol dehydrogenases that convert aketone to a hydroxyl functional group. Two such enzymes from E. coli areencoded by malate dehydrogenase (mdh) and lactate dehydrogenase (ldhA).In addition, lactate dehydrogenase from Ralstonia eutropha has beenshown to demonstrate high activities on substrates of various chainlengths such as lactate, 2-oxobutyrate, 2-oxopentanoate and2-oxoglutarate (Steinbuchel and Schlegel, Eur. J. Biochem. 130:329-334(1983)). Conversion of the oxo functionality to the hydroxyl group canalso be catalyzed by 2-ketol,3-butanediol reductase, an enzyme reportedto be found in rat and in human placenta (Suda et al., Arch. Biochem.Biophys. 176:610-620 (1976); Suda et al., Biochem. Biophys. Res. Commun.77:586-591 (1977)). All of these enzymes are good candidates for3-oxobutyraldehyde reductase. An additional candidate for these steps isthe mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) from the humanheart which has been cloned and characterized (Marks et al., J. Biol.Chem. 267:15459-15463 (1992)). This enzyme is a dehydrogenase thatoperates on a 3-hydroxyacid. Another exemplary alcohol dehydrogenasethat converts acetone to isopropanol as was shown in C. beijerinckii(Ismaiel et al., J. Bacteriol. 175:5097-5105 (1993) and T. brockii(Lamed and Zeikus, Biochem. J. 195:183-190 (1981); Peretz and Burstein,Biochemistry 28:6549-6555 (1989)). Methyl ethyl ketone (MEK) reductase,or alternatively, 2-butanol dehydrogenase, catalyzes the reduction ofMEK to form 2-butanol. Exemplary enzymes can be found in Rhodococcusruber (Kosjek et al., Biotechnol. Bioeng. 86:55-62 (2004) and Pyrococcusfuriosus (van der et al. 2001).

Protein GenBank ID GI number Organism mdh AAC76268.1 1789632 Escherichiacoli ldhA NP_415898.1 16129341 Escherichia coli ldh YP_725182.1113866693 Ralstonia eutropha bdh AAA58352.1 177198 Homo sapiens adhAAA23199.2 60592974 Clostridium beijerinckii adh P14941.1 113443Thermoanaerobacter brockii sadh CAD36475 21615553 Rhodococcus ruber adhA3288810 AAC25556 Pyrococcus furiosus

Acetoacetyl-CoA can also be reduced to 4-hydroxy,2-butanone by theCoA-dependent, alcohol forming acetoacetyl-CoA reductase. Thisintermediate is then reduced to 1,3-butanediol by 4-hydroxybutanonereductase. 4-hydroxybutanone can also be formed from 3-oxobutyraldehydeby an aldehyde reducing 3-oxobutyraldehyde reductase.

Enzymes for acetoacetyl-CoA reductase (CoA-dependent, alcohol forming)are the same as those for the alcohol-forming 3-hydroxybutyryl-CoAreductase described herein.

The enzymes for 4-hydroxybutanone reductase are the same as thosedescribed for 3-oxobutyraldehyde reductase. Additionally, a number oforganisms can catalyze the reduction of 4-hydroxy,2-butanone to1,3-butanediol, including those belonging to the genus Bacillus,Brevibacterium, Candida, and Klebsiella among others, as described byMatsuyama et al. (1).

Exemplary genes encoding enzymes that catalyze the conversion of analdehyde to alcohol (i.e., alcohol dehydrogenase or equivalentlyaldehyde reductase) include alrA encoding a medium-chain alcoholdehydrogenase for C2-C14 (Tani et al., Appl. Environ. Microbiol.66:5231-5235 (2000), ADH2 from Saccharomyces cerevisiae (Atsumi et al.,Nature 451:86-89 (2008), yqhD from E. coli which has preference formolecules longer than C3 (Sulzenbacher et al., J. Mol. Biol. 342:489-502(2004), and bdh I and bdh II from C. acetobutylicum which convertsbutyraldehyde into butanol (Walter et al., J. Bacteriol. 174:7149-7158(1992). The gene product of yqhD catalyzes the reduction ofacetaldehyde, malondialdehyde, propionaldehyde, butyraldehyde, andacrolein using NADPH as the cofactor (Perez et al., J. Biol. Chem.283:7346-7353 (2008)). ADH1 from Zymomonas mobilis has been demonstratedto have activity on a number of aldehydes including formaldehyde,acetaldehyde, propionaldehyde, butyraldehyde, and acrolein (Kinoshita etal., Appl. Microbiol. Biotechnol. 22:249-254 (1985)).

The protein sequences for each of these exemplary gene products, ifavailable, can be found using the following GenBank accession numbers:

Protein GenBank ID GI number Organism alrA BAB12273.1 9967138Acinetobacter sp. strain M-1 ADH2 NP_014032.1 6323961 Saccharomycescerevisiae yqhD NP_417484.1 16130909 Escherichia coli bdh I NP_349892.115896543 Clostridium acetobutylicum bdh II NP_349891.1 15896542Clostridium acetobutylicum adhA YP_162971.1 56552132 Zymomonas mobilis

Acetoacetyl-CoA can be converted into acetoacetate by acetoacetyl-CoAtransferase, hydrolase, or synthetase. Acetoacetate can next be reducedto 3-hydroxybutyrate by 3-hydroxybutyrate dehydrogenase and that getsfurther converted into 3-hydroxybutyraldehyde by 3-hydroxybutyratereductase. Alternatively, acetoacetate can be reduced to3-oxobutyraldehyde by acetoacetate reductase. 3-hydroxybutyryl-CoA canalso be transformed into 3-hdyroxybutyrate via 3-hydroxybutyryl-CoAtransferase, hydrolase, or synthetase.

The conversion of acetoacetyl-CoA to acetoacetate can be carried out byan acetoacetyl-CoA transferase which conserves the energy stored in theCoA-ester bond. Several exemplary transferase enzymes capable ofcatalyzing this transformation are provided below. These enzymes eithernaturally exhibit the desired acetoacetyl-CoA transferase activity orthey can be engineered via directed evolution to acceptacetetoacetyl-CoA as a substrate with increased efficiency. Suchenzymes, either naturally or following directed evolution, are alsosuitable for catalyzing the conversion of 3-hydroxybutyryl-CoA to3-hydroxybutyrate via a transferase mechanism.

Acetoacetyl-CoA:acetyl-CoA transferase naturally convertsacetoacetyl-CoA and acetate to acetoacetate and acetyl-CoA. This enzymemay also accept 3-hydroxybutyryl-CoA as a substrate or could beengineered to do so. Exemplary enzymes include the gene products ofatoAD from E. coli (Hanai et al., Appl Environ Microbiol 73:7814-7818(2007)), ctfAB from C. acetobutylicum (Jojima et al., Appl MicrobiolBiotechnol 77:1219-1224 (2008)), and ctfAB from Clostridiumsaccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem.71:58-68 (2007)). Information related to these proteins and genes isshown below.

Protein GenBank ID GI number Organism AtoA P76459.1 2492994 Escherichiacoli AtoD P76458.1 2492990 Escherichia coli CtfA NP_149326.1 15004866Clostridium acetobutylicum CtfB NP_149327.1 15004867 Clostridiumacetobutylicum CtfA AAP42564.1 31075384 Clostridiumsaccharoperbutylacetonicum CtfB AAP42565.1 31075385 Clostridiumsaccharoperbutylacetonicum

Succinyl-CoA:3-ketoacid-CoA transferase naturally converts succinate tosuccinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid.Exemplary succinyl-CoA:3:ketoacid-CoA transferases are present inHelicobacter pylori (Corthesy-Theulaz et al., J. Biol. Chem.272:25659-25667 (1997)), Bacillus subtilis (Stols et al., Protein. Expr.Purif. 53:396-403 (2007)), and Homo sapiens (Fukao et al., Genomics68:144-151 (2000); Tanaka et al., Mol. Hum. Reprod. 8:16-23 (2002)).Information related to these proteins and genes is shown below:

Protein GenBank ID GI number Organism HPAG1_0676 YP_627417 108563101Helicobacter pylori HPAG1_0677 YP_627418 108563102 Helicobacter pyloriScoA NP_391778 16080950 Bacillus subtilis ScoB NP_391777 16080949Bacillus subtilis OXCT1 NP_000427 4557817 Homo sapiens OXCT2 NP_07140311545841 Homo sapiens

Additional suitable acetoacetyl-CoA and 3-hydroxybutyryl-CoAtransferases are encoded by the gene products of cat1, cat2, and cat3 ofClostridium kluyveri. These enzymes have been shown to exhibitsuccinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA acetyltransferaseactivity, respectively (Seedorf et al., Proc. Natl. Acad. Sci. USA105:2128-2133 (2008); Sohling and Gottschalk, J Bacteriol 178:871-880(1996)). Similar CoA transferase activities are also present inTrichomonas vaginalis (van Grinsven et al., J Biol. Chem. 283:1411-1418(2008)) and Trypanosoma brucei (Riviere et al., J Biol. Chem.279:45337-45346 (2004)). Yet another transferase capable of the desiredconversions is butyryl-CoA:acetoacetate CoA-transferase. Exemplaryenzymes can be found in Fusobacterium nucleatum (Barker et al., J.Bacteriol. 152(1):201-7 (1982)), Clostridium SB4 (Barker et al., J.Biol. Chem. 253(4):1219-25 (1978)), and Clostridium acetobutylicum(Wiesenborn et al., Appl. Environ. Microbiol. 55(2):323-9 (1989)).Although specific gene sequences were not provided forbutyryl-CoA:acetoacetate CoA-transferase in these references, the genesFN0272 and FN0273 have been annotated as a butyrate-acetoacetateCoA-transferase (Kapatral et al., J Bact. 184(7) 2005-2018 (2002)).Homologs in Fusobacterium nucleatum such as FN1857 and FN1856 alsolikely have the desired acetoacetyl-CoA transferase activity. FN1857 andFN1856 are located adjacent to many other genes involved in lysinefermentation and are thus very likely to encode an acetoacetate:butyrateCoA transferase (Kreimeyer, et al., J. Biol. Chem. 282 (10) 7191-7197(2007)). Additional candidates from Porphyrmonas gingivalis andThermoanaerobacter tengcongensis can be identified in a similar fashion(Kreimeyer, et al., J Biol. Chem. 282 (10) 7191-7197 (2007)).Information related to these proteins and genes is shown below:

Protein GenBank ID GI number Organism Cat1 P38946.1 729048 Clostridiumkluyveri Cat2 P38942.2 1705614 Clostridium kluyveri Cat3 EDK35586.1146349050 Clostridium kluyveri TVAG_395550 XP_001330176 123975034Trichomonas vaginalis G3 Tb11.02.0290 XP_828352 71754875 Trypanosomabrucei FN0272 NP_603179.1 19703617 Fusobacterium nucleatum FN0273NP_603180.1 19703618 Fusobacterium nucleatum FN1857 NP_602657.1 19705162Fusobacterium nucleatum FN1856 NP_602656.1 19705161 Fusobacteriumnucleatum PG1066 NP_905281.1 34540802 Porphyromonas gingivalis W83PG1075 NP_905290.1 34540811 Porphyromonas gingivalis W83 TTE0720NP_622378.1 20807207 Thermoanaerobacter tengcongensis MB4 TTE0721NP_622379.1 20807208 Thermoanaerobacter tengcongensis MB4

Acetoacetyl-CoA can be hydrolyzed to acetoacetate by acetoacetyl-CoAhydrolase. Similarly, 3-hydroxybutyryl-CoA can be hydrolyzed to3-hydroxybutyate by 3-hydroxybutyryl-CoA hydrolase. Many CoA hydrolases(EC 3.1.2.1) have broad substrate specificity and are suitable enzymesfor these transformations either naturally or following enzymeengineering. Though the sequences were not reported, severalacetoacetyl-CoA hydrolases were identified in the cytosol andmitochondrion of the rat liver (Aragon and Lowenstein, J. Biol. Chem.258(8):4725-4733 (1983)). Additionally, an enzyme from Rattus norvegicusbrain (Robinson et al., Biochem. Biophys. Res. Commun. 71:959-965(1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. Theacot12 enzyme from the rat liver was shown to hydrolyze C2 to C6acyl-CoA molecules (Suematsu et al., Eur. J. Biochem. 268:2700-2709(2001)). Though its sequence has not been reported, the enzyme from themitochondrion of the pea leaf showed activity on acetyl-CoA,propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA, succinyl-CoA, andcrotonyl-CoA (Zeiher and Randall, Plant. Physiol. 94:20-27 (1990)).Additionally, a glutaconate CoA-transferase from Acidaminococcusfermentans was transformed by site-directed mutagenesis into an acyl-CoAhydrolase with activity on glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA(Mack and Buckel, FEBS Lett. 405:209-212 (1997)). This indicates thatthe enzymes encoding succinyl-CoA:3-ketoacid-CoA transferases andacetoacetyl-CoA:acetyl-CoA transferases can also be used as hydrolaseswith certain mutations to change their function. The acetyl-CoAhydrolase, ACH1, from S. cerevisiae represents another candidatehydrolase (Buu et al., J. Biol. Chem. 278:17203-17209 (2003)).Information related to these proteins and genes is shown below.

Protein GenBank ID GI number Organism Acot12 NP_570103.1 18543355 Rattusnorvegicus GctA CAA57199 559392 Acidaminococcus fermentans GctB CAA57200559393 Acidaminococcus fermentans ACH1 NP_009538 6319456 Saccharomycescerevisiae

Another hydrolase is the human dicarboxylic acid thioesterase, acot8,which exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA,sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., J. Biol. Chem.280:38125-38132 (2005)) and the closest E. coli homolog, tesB, which canalso hydrolyze a broad range of CoA thioesters (Naggert et al., J. Biol.Chem. 266:11044-11050 (1991)) including 3-hydroxybutyryl-CoA (Tseng etal., Appl. Environ. Microbiol. 75(10):3137-3145 (2009)). A similarenzyme has also been characterized in the rat liver (Deana, Biochem.Int. 26:767-773 (1992)). Other potential E. coli thioester hydrolasesinclude the gene products of tesA (Bonner and Bloch, J. Biol. Chem.247:3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS Microbiol. Rev.29:263-279 (2005); Zhuang et al., FEBS Lett. 516:161-163 (2002)), paaI(Song et al., J. Biol. Chem. 281:11028-11038 (2006)), and ybdB (Leduc etal., J. Bacteriol. 189:7112-7126 (2007)). Information related to theseproteins and genes is shown below:

Protein GenBank ID GI number Organism Acot8 CAA15502 3191970 Homosapiens TesB NP_414986 16128437 Escherichia coli Acot8 NP_57011251036669 Rattus norvegicus TesA NP_415027 16128478 Escherichia coli YbgCNP_415264 16128711 Escherichia coli PaaI NP_415914 16129357 Escherichiacoli YbdB NP_415129 16128580 Escherichia coli

Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA hydrolasewhich has been described to efficiently catalyze the conversion of3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate during valinedegradation (Shimomura et al., J Biol. Chem. 269:14248-14253 (1994)).Genes encoding this enzyme include hibch of Rattus norvegicus (Shimomuraet al., supra (1994); Shimomura et al., Methods Enzymol. 324:229-240(2000)) and Homo sapiens (Shimomura et al., supra (1994). Candidategenes by sequence homology include hibch of Saccharomyces cerevisiae andBC 2292 of Bacillus cereus. BC_2292 was shown to demonstrate3-hydroxybutyryl-CoA hydrolase activity and function as part of apathway for 3-hydroxybutyrate synthesis when engineered into Escherichiacoli (Lee et al., Appl. Microbiol. Biotechnol. 79:633-641 (2008)).Information related to these proteins and genes is shown below:

Protein GenBank ID GI number Organism Hibch Q5XIE6.2 146324906 Rattusnorvegicus Hibch Q6NVY1.2 146324905 Homo sapiens Hibch P28817.2 2506374Saccharomyces cerevisiae BC_2292 AP09256 29895975 Bacillus cereus ATCC14579

An alternative method for removing the CoA moiety from acetoacetyl-CoAor 3-hydroxybutyryl-CoA is to apply a pair of enzymes such as aphosphate-transferring acyltransferase and a kinase to impartacetoacetyl-CoA or 3-hydroxybutyryl-CoA synthetase activity. Thisactivity enables the net hydrolysis of the CoA-ester of either moleculewith the simultaneous generation of ATP. For example, the butyratekinase (buk)/phosphotransbutyrylase (ptb) system from Clostridiumacetobutylicum has been successfully applied to remove the CoA groupfrom 3-hydroxybutyryl-CoA when functioning as part of a pathway for3-hydroxybutyrate synthesis (Tseng et al., Appl. Environ. Microbiol.75(10):3137-3145 (2009)). Specifically, the ptb gene from C.acetobutylicum encodes an enzyme that can convert an acyl-CoA into anacyl-phosphate (Walter et al. Gene 134(1): p. 107-11 (1993)); Huang etal. J Mol. Microbiol Biotechnol 2(1): p. 33-38 (2000). Additional ptbgenes can be found in butyrate-producing bacterium L2-50 (Louis et al.J. Bacteriol. 186:2099-2106 (2004)) and Bacillus megaterium (Vazquez etal. Curr. Microbiol 42:345-349 (2001)). Additional exemplaryphosphate-transferring acyltransferases include phosphotransacetylase,encoded by pta. The pta gene from E. coli encodes an enzyme that canconvert acetyl-CoA into acetyl-phosphate, and vice versa (Suzuki, T.Biochim. Biophys. Acta 191:559-569 (1969)). This enzyme can also utilizepropionyl-CoA instead of acetyl-CoA forming propionate in the process(Hesslinger et al. Mol. Microbiol 27:477-492 (1998)). Informationrelated to these proteins and genes is shown below:

Protein GenBank ID GI number Organism Pta NP_416800.1 16130232Escherichia coli Ptb NP_349676 15896327 Clostridium acetobutylicum PtbAAR19757.1 38425288 butyrate-producing bacterium L2-50 Ptb CAC07932.110046659 Bacillus megaterium

Exemplary kinases include the E. coli acetate kinase, encoded by ackA(Skarstedt and Silverstein J. Biol. Chem. 251:6775-6783 (1976)), the C.acetobutylicum butyrate kinases, encoded by buk1 and buk2 ((Walter etal. Gene 134(1):107-111 (1993); Huang et al. J Mol Microbiol Biotechnol2(1):33-38 (2000)), and the E. coli gamma-glutamyl kinase, encoded byproB (Smith et al. J. Bacteriol. 157:545-551 (1984)). These enzymesphosphorylate acetate, butyrate, and glutamate, respectively. The ackAgene product from E. coli also phosphorylates propionate (Hesslinger etal. Mol. Microbiol 27:477-492 (1998)). Information related to theseproteins and genes is shown below:

Protein GenBank ID GI number Organism AckA NP_416799.1 16130231Escherichia coli Buk1 NP_349675 15896326 Clostridium acetobutylicum Buk2Q97II1 20137415 Clostridium acetobutylicum ProB NP_414777.1 16128228Escherichia coli

The hydrolysis of acetoacetyl-CoA or 3-hydroxybutyryl-CoA canalternatively be carried out by a single enzyme or enzyme complex thatexhibits acetoacetyl-CoA or 3-hydroxybutyryl-CoA synthetase activity.This activity enables the net hydrolysis of the CoA-ester of eithermolecule, and in some cases, results in the simultaneous generation ofATP. For example, the product of the LSC1 and LSC2 genes of S.cerevisiae and the sucC and sucD genes of E. coli naturally form asuccinyl-CoA synthetase complex that catalyzes the formation ofsuccinyl-CoA from succinate with the concomitant consumption of one ATP,a reaction which is reversible in vivo (Gruys et al., U.S. Pat. No.5,958,745, filed Sep. 28, 1999). Information related to these proteinsand genes is shown below:

Protein GenBank ID GI number Organism SucC NP_415256.1 16128703Escherichia coli SucD AAC73823.1 1786949 Escherichia coli LSC1 NP_0147856324716 Saccharomyces cerevisiae LSC2 NP_011760 6321683 Saccharomycescerevisiae

Additional exemplary CoA-ligases include the rat dicarboxylate-CoAligase for which the sequence is yet uncharacterized (Vamecq et al.,Biochemical J. 230:683-693 (1985)), either of the two characterizedphenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et al.,Biochem. J. 395:147-155 (2005); Wang et al., Biochem Biophy Res Commun360(2):453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonasputida (Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)),and the 6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Bower etal., J Bacteriol. 178(14):4122-4130 (1996)). Additional candidateenzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa etal., Biochim. Biophys. Acta 1779:414-419 (2008)) and Homo sapiens(Ohgami et al., Biochem. Pharmacol. 65:989-994 (2003)), which naturallycatalyze the ATP-dependant conversion of acetoacetate intoacetoacetyl-CoA. 4-Hydroxybutyryl-CoA synthetase activity has beendemonstrated in Metallosphaera sedula (Berg et al., Science318:1782-1786 (2007)). This function has been tentatively assigned tothe Msed 1422 gene. Information related to these proteins and genes isshown below:

Protein GenBank ID GI number Organism Phl CAJ15517.1 77019264Penicillium chrysogenum PhlB ABS19624.1 152002983 Penicilliumchrysogenum PaaF AAC24333.2 22711873 Pseudomonas putida BioW NP_390902.250812281 Bacillus subtilis AACS NP_084486.1 21313520 Mus musculus AACSNP_076417.2 31982927 Homo sapiens Msed_1422 YP_001191504 146304188Metallosphaera sedula

ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is anothercandidate enzyme that couples the conversion of acyl-CoA esters to theircorresponding acids with the concurrent synthesis of ATP. Severalenzymes with broad substrate specificities have been described in theliterature. ACD I from Archaeoglobus fulgidus, encoded by AF1211, wasshown to operate on a variety of linear and branched-chain substratesincluding acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate,butyrate, isobutyryate, isovalerate, succinate, fumarate, phenylacetate,indoleacetate (Musfeldt et al., J. Bacteriol. 184:636-644 (2002)). Theenzyme from Haloarcula marismortui (annotated as a succinyl-CoAsynthetase) accepts propionate, butyrate, and branched-chain acids(isovalerate and isobutyrate) as substrates, and was shown to operate inthe forward and reverse directions (Brasen et al., Arch. Microbiol.182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophiliccrenarchaeon Pyrobaculum aerophilum showed the broadest substrate rangeof all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA(preferred substrate) and phenylacetyl-CoA (Brasen et al., supra(2004)). The enzymes from A. fulgidus, H. marismortui and P. aerophilumhave all been cloned, functionally expressed, and characterized in E.coli (Musfeldt et al., supra; Brasen et al., supra (2004)). Informationrelated to these proteins and genes is shown below:

Protein GenBank ID GI number Organism AF1211 NP_070039.1 11498810Archaeoglobus fulgidus DSM 4304 scs YP_135572.1 55377722 Haloarculamarismortui ATCC 43049 PAE3250 NP_560604.1 18313937 Pyrobaculumaerophilum str. IM2

The conversion of 3-hydroxybutyrate to 3-hydroxybutyraldehyde can becarried out by a 3-hydroxybutyrate reductase. Similarly, the conversionof acetoacetate to acetoacetaldehyde can be carried out by anacetoacetate reductase. A suitable enzyme for these transformations isthe aryl-aldehyde dehydrogenase, or equivalently a carboxylic acidreductase, from Nocardia iowensis. Carboxylic acid reductase catalyzesthe magnesium, ATP and NADPH-dependent reduction of carboxylic acids totheir corresponding aldehydes (Venkitasubramanian et al., J. Biol. Chem.282:478-485 (2007)). This enzyme, encoded by car, was cloned andfunctionally expressed in E. coli (Venkitasubramanian et al., J. Biol.Chem. 282:478-485 (2007)). Expression of the npt gene product improvedactivity of the enzyme via post-transcriptional modification. The nptgene encodes a specific phosphopantetheine transferase (PPTase) thatconverts the inactive apo-enzyme to the active holo-enzyme. The naturalsubstrate of this enzyme is vanillic acid, and the enzyme exhibits broadacceptance of aromatic and aliphatic substrates (Venkitasubramanian etal., in Biocatalysis in the Pharmaceutical and Biotechnology Industires,ed. R. N. Patel, Chapter 15, pp. 425-440, CRC Press LLC, Boca Raton,Fla. (2006)). Information related to these proteins and genes is shownbelow:

Protein GI Number genbank ID Organism Car 40796035 AAR91681.1 Nocardiaiowensis (sp. NRRL 5646) Npt 114848891 ABI83656.1 Nocardia iowensis (sp.NRRL 5646)

Additional car and npt genes can be identified based on sequencehomology.

Protein GENBANK ID GI Number Organism fadD9 YP_978699.1 121638475Mycobacterium bovis BCG BCG_2812c YP_978898.1 121638674 Mycobacteriumbovis BCG nfa20150 YP_118225.1 54023983 Nocardia farcinica IFM 10152nfa40540 YP_120266.1 54026024 Nocardia farcinica IFM 10152 SGR_6790YP_001828302.1 182440583 Streptomyces griseus subsp. griseus NBRC 13350SGR_665 YP_001822177.1 182434458 Streptomyces griseus subsp. griseusNBRC 13350 MSMEG_2956 YP_887275.1 118473501 Mycobacterium smegmatis MC2155 MSMEG_5739 YP_889972.1 118469671 Mycobacterium smegmatis MC2 155MSMEG_2648 YP_886985.1 118471293 Mycobacterium smegmatis MC2 155MAP1040c NP_959974.1 41407138 Mycobacterium avium subsp.paratuberculosis K-10 MAP2899c NP_961833.1 41408997 Mycobacterium aviumsubsp. paratuberculosis K-10 MMAR_2117 YP_001850422.1 183982131Mycobacterium marinum M MMAR_2936 YP_001851230.1 183982939 Mycobacteriummarinum M MMAR_1916 YP_001850220.1 183981929 Mycobacterium marinum MTpauDRAFT_33060 ZP_04027864.1 227980601 Tsukamurella paurometabola DSM20162 TpauDRAFT_20920 ZP_04026660.1 227979396 Tsukamurella paurometabolaDSM 20162 CPCC7001_1320 ZP_05045132.1 254431429 Cyanobium PCC7001DDBDRAFT_0187729 XP_636931.1 66806417 Dictyostelium discoideum AX4

An additional enzyme candidate found in Streptomyces griseus is encodedby the griC and griD genes. This enzyme is believed to convert3-amino-4-hydroxybenzoic acid to 3-amino-4-hydroxybenzaldehyde asdeletion of either griC or griD led to accumulation of extracellular3-acetylamino-4-hydroxybenzoic acid, a shunt product of3-amino-4-hydroxybenzoic acid metabolism (Suzuki, et al., J. Antibiot.60(6):380-387 (2007)). Co-expression of griC and griD with SGR_665, anenzyme similar in sequence to the Nocardia iowensis npt, can bebeneficial. Information related to these proteins and genes is shownbelow:

Protein GI number genbank ID Organism grzC 182438036 YP_001825755.1Streptomyces griseus subsp. griseus NBRC 13350 griD 182438037YP_001825756.1 Streptomyces griseus subsp. griseus NBRC 13350

An enzyme with similar characteristics, alpha-aminoadipate reductase(AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in somefungal species. This enzyme naturally reduces alpha-aminoadipate toalpha-aminoadipate semialdehyde. The carboxyl group is first activatedthrough the ATP-dependent formation of an adenylate that is then reducedby NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizesmagnesium and requires activation by a PPTase. Enzyme candidates for AARand its corresponding PPTase are found in Saccharomyces cerevisiae(Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al.,Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe(Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S. pombeexhibited significant activity when expressed in E. coli (Guo et al.,Yeast 21:1279-1288 (2004)). The AAR from Penicillium chrysogenum acceptsS-carboxymethyl-L-cysteine as an alternate substrate, but did not reactwith adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J.Biol. Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenumPPTase has not been identified to date. Information related to theseproteins and genes is shown below:

Protein GI number genbank ID Organism LYS2 171867 AAA34747.1Saccharomyces cerevisiae LYS5 1708896 P50113.1 Saccharomyces cerevisiaeLYS2 2853226 AAC02241.1 Candida albicans LYS5 28136195 AAO26020.1Candida albicans Lys1p 13124791 P40976.3 Schizosaccharomyces pombe Lys7p1723561 Q10474.1 Schizosaccharomyces pombe Lys2 3282044 CAA74300.1Penicillium chrysogenum

Any of these CAR or CAR-like enzymes can exhibit 3-hydroxybutyrate oracetoacetate reductase activity or can be engineered to do so.

The requisite 3-hydroxybutyrate dehydrogenase catalyzes the reduction ofacetoacetate to form 3-hydroxybutyrate. Exemplary enzymes can be foundin Rhodococcus ruber (Kosjek et al., Biotechnol Bioeng. 86:55-62 (2004))and Pyrococcus furiosus (van der et al., Eur. J. Biochem. 268:3062-3068(2001)). Additional secondary alcohol dehydrogenase enzymes capable ofthis transformation include adh from C. beijerinckii (Hanai et al., ApplEnviron Microbiol 73:7814-7818 (2007); Jojima et al., Appl MicrobiolBiotechnol 77:1219-1224 (2008)) and adh from Thermoanaerobacter brockii(Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007); Peretz etal., Anaerobe 3:259-270 (1997)). The cloning of the bdhA gene fromRhizobium (Sinorhizobium) Meliloti into E. coli conferred the ability toutilize 3-hydroxybutyrate as a carbon source (Aneja and Charles, J.Bacteriol. 181(3):849-857 (1999)). Additional 3-hydroxybutyratedehydrogenase can be found in Pseudomonas fragi (Ito et al., J. Mol.Biol. 355(4) 722-733 (2006)) and Ralstonia pickettii (Takanashi et al.,Antonie van Leeuwenoek, 95(3):249-262 (2009)). Information related tothese proteins and genes is shown below:

Protein Genbank ID GI Number Organism Sadh CAD36475 21615553 Rhodococcusrubber AdhA AAC25556 3288810 Pyrococcus furiosus Adh P14941.1 113443Thermoanaerobobacter brockii Adh AAA23199.2 60592974 Clostridiumbeijerinckii BdhA NP_437676.1 16264884 Rhizobium (Sinorhizobium)Meliloti PRK13394 BAD86668.1 57506672 Pseudomonas fragi Bdh1 BAE72684.184570594 Ralstonia pickettii Bdh2 BAE72685.1 84570596 Ralstoniapickettii Bdh3 BAF91602.1 158937170 Ralstonia pickettii

Example II Isopropanol Synthesis Pathway

This Example shows how isopropanol production was achieved inrecombinant E. coli following expression of two heterologous genes fromC. acetobutylicum (thl and adc encoding acetoacetyl-CoA thiolase andacetoacetate decarboxylase, respectively) and one from C. beijerinckii(adh encoding a secondary alcohol dehydrogenase), along with theincreased expression of the native atoA and atoD genes which encodeacetoacetyl-CoA:acetate:CoA transferase activity (Hanai et al., Appl.Environ. Microbiol. 73:7814-7818 (2007)). The acetoacetyl-CoA thiolase(AtoB) enzymes are described herein above.

The conversion of acetoacetyl-CoA to acetoacetate or of4-hydroxybutyryl-CoA to 4-hydroxybutyrate can be carried out by anacetoacetyl-CoA transferase or 4-hydroxybutyryl-CoA transferase,respectively. These enzymes conserve the energy stored in the CoA-esterbonds of acetoacetyl-CoA and 4-hydroxybutyryl-CoA. Many transferaseshave broad specificity and thus may utilize CoA acceptors as diverse asacetate, succinate, propionate, butyrate, 2-methylacetoacetate,3-ketohexanoate, 3-ketopentanoate, valerate, crotonate,3-mercaptopropionate, propionate, vinylacetate, butyrate, among others.Acetoacetyl-CoA transferase catalyzes the conversion of acetoacetyl-CoAto acetoacetate while transferring the CoA moiety to a CoA acceptormolecule. Several exemplary transferase enzymes capable of catalyzingthis transformation are provided below. These enzymes either naturallyexhibit the desired acetoacetyl-CoA transferase activity or they can beengineered via directed evolution to accept acetoacetyl-CoA as asubstrate with increased efficiency. Such enzymes, either naturally orfollowing directed evolution, are also suitable for catalyzing theconversion of 4-hydroxybutyryl-CoA to 4-hydroxybutyrate via atransferase mechanism.

In one embodiment an exemplary acetoacetyl-CoA transferase isacetoacetyl-CoA:acetate-CoA transferase. This enzyme naturally convertsacetate to acetyl-CoA while converting acetoacetyl-CoA to acetoacetate.In another embodiment, a succinyl-CoA:3-ketoacid CoA transferase (SCOT)catalyzes the conversion of the 3-ketoacyl-CoA, acetoacetyl-CoA, to the3-ketoacid, acetoacetate.

Acetoacetyl-CoA:acetyl-CoA transferase naturally convertsacetoacetyl-CoA and acetate to acetoacetate and acetyl-CoA. This enzymecan also accept 3-hydroxybutyryl-CoA as a substrate or could beengineered to do so. Exemplary enzymes include the gene products ofatoAD from E. coli (Hanai et al., Appl Environ Microbiol 73:7814-7818(2007)), ctfAB from C. acetobutylicum (Jojima et al., Appl MicrobiolBiotechnol 77:1219-1224 (2008)), and ctfAB from Clostridiumsaccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem.71:58-68 (2007)). Information related to these proteins and genes isshown below:

Protein GENBANK ID GI NUMBER ORGANISM AtoA P76459.1 2492994 Escherichiacoli AtoD P76458.1 2492990 Escherichia coli CtfA NP_149326.1 15004866Clostridium acetobutylicum CtfB NP_149327.1 15004867 Clostridiumacetobutylicum CtfA AAP42564.1 31075384 Clostridiumsaccharoperbutylacetonicum CtfB AAP42565.1 31075385 Clostridiumsaccharoperbutylacetonicum

Succinyl-CoA:3-ketoacid-CoA transferase naturally converts succinate tosuccinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid.Exemplary succinyl-CoA:3:ketoacid-CoA transferases are present inHelicobacter pylori (Corthesy-Theulaz et al., J. Biol. Chem.272:25659-25667 (1997)), Bacillus subtilis (Stols et al., Protein. Expr.Purif. 53:396-403 (2007)), and Homo sapiens (Fukao et al., Genomics68:144-151 (2000); Tanaka et al., Mol. Hum. Reprod. 8:16-23 (2002)).Information related to these proteins and genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM HPAG1_0676 YP_627417 108563101Helicobacter pylori HPAG1_0677 YP_627418 108563102 Helicobacter pyloriScoA NP_391778 16080950 Bacillus subtilis ScoB NP_391777 16080949Bacillus subtilis OXCT1 NP_000427 4557817 Homo sapiens OXCT2 NP_07140311545841 Homo sapiens

Additional suitable acetoacetyl-CoA and 4-hydroxybutyryl-CoAtransferases are encoded by the gene products of cat1, cat2, and cat3 ofClostridium kluyveri. These enzymes have been shown to exhibitsuccinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferaseactivity, respectively (Seedorf et al., Proc. Natl. Acad. Sci. USA105:2128-2133 (2008); Sohling and Gottschalk, J Bacteriol 178:871-880(1996)). Similar CoA transferase activities are also present inTrichomonas vaginalis (van Grinsven et al., J Biol. Chem. 283:1411-1418(2008)) and Trypanosoma brucei (Riviere et al., J. Biol. Chem.279:45337-45346 (2004)). Yet another transferase capable of the desiredconversions is butyryl-CoA:acetoacetate CoA-transferase. Exemplaryenzymes can be found in Fusobacterium nucleatum (Barker et al., J.Bacteriol. 152(1):201-7 (1982)), Clostridium SB4 (Barker et al., J Biol.Chem. 253(4):1219-25 (1978)), and Clostridium acetobutylicum (Wiesenbornet al., Appl. Environ. Microbiol. 55(2):323-9 (1989)). Although specificgene sequences were not provided for butyryl-CoA:acetoacetateCoA-transferase in these references, the genes FN0272 and FN0273 havebeen annotated as a butyrate-acetoacetate CoA-transferase (Kapatral etal., J. Bact. 184(7) 2005-2018 (2002)). Homologs in Fusobacteriumnucleatum such as FN1857 and FN1856 also likely have the desiredacetoacetyl-CoA transferase activity. FN1857 and FN1856 are locatedadjacent to many other genes involved in lysine fermentation and arethus very likely to encode an acetoacetate:butyrate CoA transferase(Kreimeyer, et al., J. Biol. Chem. 282 (10) 7191-7197 (2007)).Additional candidates from Porphyrmonas gingivalis andThermoanaerobacter tengcongensis can be identified in a similar fashion(Kreimeyer, et al., J. Biol. Chem. 282 (10) 7191-7197 (2007)).Information related to these proteins and genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM Cat1 P38946.1 729048 Clostridiumkluyveri Cat2 P38942.2 1705614 Clostridium kluyveri Cat3 EDK35586.1146349050 Clostridium kluyveri TVAG_395550 XP_001330176 123975034Trichomonas vaginalis G3 Tb11.02.0290 XP_828352 71754875 Trypanosomabrucei FN0272 NP_603179.1 19703617 Fusobacterium nucleatum FN0273NP_603180.1 19703618 Fusobacterium nucleatum FN1857 NP_602657.1 19705162Fusobacterium nucleatum FN1856 NP_602656.1 19705161 Fusobacteriumnucleatum PG1066 NP_905281.1 34540802 Porphyromonas gingivalis W83PG1075 NP_905290.1 34540811 Porphyromonas gingivalis W83 TTE0720NP_622378.1 20807207 Thermoanaerobacter tengcongensis MB4 TTE0721NP_622379.1 20807208 Thermoanaerobacter tengcongensis MB4

Acetoacetyl-CoA can be hydrolyzed to acetoacetate by acetoacetyl-CoAhydrolase. Similarly, 4-hydroxybutyryl-CoA can be hydrolyzed to4-hydroxybutyate by 4-hydroxybutyryl-CoA hydrolase. Many CoA hydrolases(EC 3.1.2.1) have broad substrate specificity and are suitable enzymesfor these transformations either naturally or following enzymeengineering. Though the sequences were not reported, severalacetoacetyl-CoA hydrolases were identified in the cytosol andmitochondrion of the rat liver (Aragon and Lowenstein, J. Biol. Chem.258(8):4725-4733 (1983)). Additionally, an enzyme from Rattus norvegicusbrain (Robinson et al., Biochem. Biophys. Res. Commun. 71:959-965(1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. Theacot12 enzyme from the rat liver was shown to hydrolyze C2 to C6acyl-CoA molecules (Suematsu et al., Eur. J. Biochem. 268:2700-2709(2001)). Though its sequence has not been reported, the enzyme from themitochondrion of the pea leaf showed activity on acetyl-CoA,propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA, succinyl-CoA, andcrotonyl-CoA (Zeiher and Randall, Plant. Physiol. 94:20-27 (1990)).Additionally, a glutaconate CoA-transferase from Acidaminococcusfermentans was transformed by site-directed mutagenesis into an acyl-CoAhydrolase with activity on glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA(Mack and Buckel, FEBS Lett. 405:209-212 (1997)). This indicates thatthe enzymes encoding acetoacetyl-CoA transferases and4-hydroxybutyryl-CoA transferases can also be used as hydrolases withcertain mutations to change their function. The acetyl-CoA hydrolase,ACH1, from S. cerevisiae represents another candidate hydrolase (Buu etal., J. Biol. Chem. 278:17203-17209 (2003)). Information related tothese proteins and genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM Acot12 NP_570103.1 18543355 Rattusnorvegicus GctA CAA57199 559392 Acidaminococcus fermentans GctB CAA57200559393 Acidaminococcus fermentans ACH1 NP_009538 6319456 Saccharomycescerevisiae

Another candidate hydrolase is the human dicarboxylic acid thioesterase,acot8, which exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA,sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., J. Biol. Chem.280:38125-38132 (2005)) and the closest E. coli homolog, tesB, which canalso hydrolyze a broad range of CoA thioesters (Naggert et al., J. Biol.Chem. 266:11044-11050 (1991)) including 3-hydroxybutyryl-CoA (Tseng etal., Appl. Environ. Microbiol. 75(10):3137-3145 (2009)). A similarenzyme has also been characterized in the rat liver (Deana, Biochem.Int. 26:767-773 (1992)). Other potential E. coli thioester hydrolasesinclude the gene products of tesA (Bonner and Bloch, J. Biol. Chem.247:3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS Microbiol. Rev.29:263-279 (2005); Zhuang et al., FEBS Lett. 516:161-163 (2002)), paaI(Song et al., J. Biol. Chem. 281:11028-11038 (2006)), and ybdB (Leduc etal., J. Bacteriol. 189:7112-7126 (2007)). Information related to theseproteins and genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM Acot8 CAA15502 3191970 Homosapiens TesB NP_414986 16128437 Escherichia coli Acot8 NP_57011251036669 Rattus norvegicus TesA NP_415027 16128478 Escherichia coli YbgCNP_415264 16128711 Escherichia coli PaaI NP_415914 16129357 Escherichiacoli YbdB NP_415129 16128580 Escherichia coli

Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA hydrolasewhich has been described to efficiently catalyze the conversion of3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate during valinedegradation (Shimomura et al., J. Biol. Chem. 269:14248-14253 (1994)).Genes encoding this enzyme include hibch of Rattus norvegicus (Shimomuraet al., supra (1994); Shimomura et al., Methods Enzymol. 324:229-240(2000)) and Homo sapiens (Shimomura et al., supra (1994). Candidategenes by sequence homology include hibch of Saccharomyces cerevisiae andBC_2292 of Bacillus cereus. BC_2292 was shown to demonstrate3-hydroxybutyryl-CoA hydrolase activity and function as part of apathway for 3-hydroxybutyrate synthesis when engineered into Escherichiacoli (Lee et al., Appl. Microbiol. Biotechnol. 79:633-641 (2008)).Information related to these proteins and genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM Hibch Q5XIE6.2 146324906 Rattusnorvegicus Hibch Q6NVY1.2 146324905 Homo sapiens Hibch P28817.2 2506374Saccharomyces cerevisiae BC_2292 AP09256 29895975 Bacillus cereus ATCC14579

The hydrolysis of acetoacetyl-CoA or 4-hydroxybutyryl-CoA canalternatively be carried out by a single enzyme or enzyme complex thatexhibits acetoacetyl-CoA or 4-hydroxybutyryl-CoA synthetase activity.This activity enables the net hydrolysis of the CoA-ester of eithermolecule, and in some cases, results in the simultaneous generation ofATP. For example, the product of the LSC1 and LSC2 genes of S.cerevisiae and the sucC and sucD genes of E. coli naturally form asuccinyl-CoA synthetase complex that catalyzes the formation ofsuccinyl-CoA from succinate with the concomitant consumption of one ATP,a reaction which is reversible in vivo (Gruys et al., U.S. Pat. No.5,958,745, filed Sep. 28, 1999). Information related to these proteinsand genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM SucC NP_415256.1 16128703Escherichia coli SucD AAC73823.1 1786949 Escherichia coli LSC1 NP_0147856324716 Saccharomyces cerevisiae LSC2 NP_011760 6321683 Saccharomycescerevisiae

Additional exemplary CoA-ligases include the rat dicarboxylate-CoAligase for which the sequence is yet uncharacterized (Vamecq et al.,Biochemical J. 230:683-693 (1985)), either of the two characterizedphenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et al.,Biochem. J. 395:147-155 (2005); Wang et al., Biochem Biophy Res Commun360(2):453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonasputida (Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)),and the 6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Bower etal., J. Bacteriol. 178(14):4122-4130 (1996)). Additional candidateenzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa etal., Biochim. Biophys. Acta 1779:414-419 (2008)) and Homo sapiens(Ohgami et al., Biochem. Pharmacol. 65:989-994 (2003)), which naturallycatalyze the ATP-dependant conversion of acetoacetate intoacetoacetyl-CoA. 4-Hydroxybutyryl-CoA synthetase activity has beendemonstrated in Metallosphaera sedula (Berg et al., Science318:1782-1786 (2007)). This function has been tentatively assigned tothe Msed 1422 gene. Information related to these proteins and genes isshown below:

Protein GENBANK ID GI NUMBER ORGANISM Phl CAJ15517.1 77019264Penicillium chrysogenum PhlB ABS19624.1 152002983 Penicilliumchrysogenum PaaF AAC24333.2 22711873 Pseudomonas putida BioW NP_390902.250812281 Bacillus subtilis AACS NP_084486.1 21313520 Mus musculus AACSNP_076417.2 31982927 Homo sapiens Msed_1422 YP_001191504 146304188Metallosphaera sedula

ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is anothercandidate enzyme that couples the conversion of acyl-CoA esters to theircorresponding acids with the concurrent synthesis of ATP. Severalenzymes with broad substrate specificities have been described in theliterature. ACD I from Archaeoglobus fulgidus, encoded by AF1211, wasshown to operate on a variety of linear and branched-chain substratesincluding acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate,butyrate, isobutyrate, isovalerate, succinate, fumarate, phenylacetate,indoleacetate (Musfeldt et al., J. Bacteriol. 184:636-644 (2002)). Theenzyme from Haloarcula marismortui (annotated as a succinyl-CoAsynthetase) accepts propionate, butyrate, and branched-chain acids(isovalerate and isobutyrate) as substrates, and was shown to operate inthe forward and reverse directions (Brasen et al., Arch. Microbiol.182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophiliccrenarchaeon Pyrobaculum aerophilum showed the broadest substrate rangeof all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA(preferred substrate) and phenylacetyl-CoA (Brasen et al., supra(2004)). The enzymes from A. fulgidus, H. marismortui and P. aerophilumhave all been cloned, functionally expressed, and characterized in E.coli (Musfeldt et al., supra; Brasen et al., supra (2004)). Informationrelated to these proteins and genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM AF1211 NP_070039.1 11498810Archaeoglobus fulgidus DSM 4304 Scs YP_135572.1 55377722 Haloarculamarismortui ATCC 43049 PAE3250 NP_560604.1 18313937 Pyrobaculumaerophilum str. IM2

An alternative method for removing the CoA moiety from acetoacetyl-CoAor 4-hydroxybutyryl-CoA is to apply a pair of enzymes such as aphosphate-transferring acyltransferase and a kinase to impartacetoacetyl-CoA or 4-hydroxybutyryl-CoA synthetase activity. Exemplarynames for these enzymes includephosphotrans-4-hydroxybutyrylase/4-hydroxybutyrate kinase, which canremove the CoA moiety from 4-hydroxybutyryl-CoA, andphosphotransacetoacetylase/acetoacetate kinase which can remove the CoAmoiety from acetoacetyl-CoA. This general activity enables the nethydrolysis of the CoA-ester of either molecule with the simultaneousgeneration of ATP. For example, the butyrate kinase(buk)/phosphotransbutyrylase (ptb) system from Clostridiumacetobutylicum has been successfully applied to remove the CoA groupfrom 3-hydroxybutyryl-CoA when functioning as part of a pathway for3-hydroxybutyrate synthesis (Tseng et al., Appl. Environ. Microbiol.75(10):3137-3145 (2009)). Specifically, the ptb gene from C.acetobutylicum encodes an enzyme that can convert an acyl-CoA into anacyl-phosphate (Walter et al. Gene 134(1): p. 107-11 (1993)); Huang etal. J Mol Microbiol Biotechnol 2(1): p. 33-38 (2000). Additional ptbgenes can be found in butyrate-producing bacterium L2-50 (Louis et al.J. Bacteriol. 186:2099-2106 (2004)) and Bacillus megaterium (Vazquez etal. Curr. Microbiol 42:345-349 (2001)). Additional exemplaryphosphate-transferring acyltransferases include phosphotransacetylase,encoded by pta. The pta gene from E. coli encodes an enzyme that canconvert acetyl-CoA into acetyl-phosphate, and vice versa (Suzuki, T.Biochim. Biophys. Acta 191:559-569 (1969)). This enzyme can also utilizepropionyl-CoA instead of acetyl-CoA forming propionate in the process(Hesslinger et al. Mol. Microbiol 27:477-492 (1998)). Informationrelated to these proteins and genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM Pta NP_416800.1 16130232Escherichia coli Ptb NP_349676 15896327 Clostridium acetobutylicum PtbAAR19757.1 38425288 butyrate-producing bacterium L2-50 Ptb CAC07932.110046659 Bacillus megaterium

Exemplary kinases include the E. coli acetate kinase, encoded by ackA(Skarstedt and Silverstein J. Biol. Chem. 251:6775-6783 (1976)), the C.acetobutylicum butyrate kinases, encoded by buk1 and buk2 ((Walter etal. Gene 134(1):107-111 (1993); Huang et al. J Mol Microbiol Biotechnol2(1):33-38 (2000)), and the E. coli gamma-glutamyl kinase, encoded byproB (Smith et al. J. Bacteriol. 157:545-551 (1984)). These enzymesphosphorylate acetate, butyrate, and glutamate, respectively. The ackAgene product from E. coli also phosphorylates propionate (Hesslinger etal. Mol. Microbiol 27:477-492 (1998)). Information related to theseproteins and genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM AckA NP_416799.1 16130231Escherichia coli Buk1 NP_349675 15896326 Clostridium acetobutylicum Buk2Q97II1 20137415 Clostridium acetobutylicum ProB NP_414777.1 16128228Escherichia coli

Acetoacetate decarboxylase converts acetoacetate into carbon dioxide andacetone. Exemplary acetoacetate decarboxylase enzymes are encoded by thegene products of adc from C. acetobutylicum (Petersen and Bennett, Appl.Environ. Microbiol. 56:3491-3498 (1990) and adc from Clostridiumsaccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol. Biochem.71:58-68 (2007)). The enzyme from C. beijerinkii can be inferred fromsequence similarity.

Protein GenBank ID GI Number Organism Adc NP_149328.1 15004868Clostridium acetobutylicum Adc AAP42566.1 31075386 Clostridiumsaccharoperbutylacetonicum Adc YP_001310906.1 150018652 Clostridiumbeijerinckii

The final step in the isopropanol synthesis pathway involves thereduction of acetone to isopropanol. Exemplary alcohol dehydrogenaseenzymes capable of this transformation include adh from C. beijerinckii(Jojima et al., Appl. Microbiol. Biotechnol. 77:1219-1224 (2008); Hanaiet al., Appl. Environ. Microbiol. 73:7814-7818 (2007) and adh fromThermoanaerobacter brockii (Hanai et al., supra; Peretz et al., Anaerobe3:259-270 (1997)). Additional characterized enzymes include alcoholdehydrogenases from Ralstonia eutropha (formerly Alcaligenes eutrophus)(Steinbuchel and Schlegel, Eur. J. Biochem. 141:555-564 (1984) andPhytomonas species (Uttaro and Opperdoes, Mol. Biochen. Parasitol. 85:213-219 (1997)).

Protein GenBank ID GI Number Organism Adh P14941.1 113443Thermoanaerobobacter brockii Adh AAA23199.2 60592974 Clostridiumbeijerinckii Adh YP_299391.1 73539024 Ralstonia eutropha iPDH AAP39869.131322946 Phtomonas sp.

Example III 4-Hydroxybutyrate Synthesis Pathway

This Example shows further enzymes that can be used in a4-hydroxybutyrate pathway. The genes for the first enzyme,acetoacetyl-CoA thiolase are described herein above.

Exemplary 3-hydroxyacyl dehydrogenases which convert acetoacetyl-CoA to3-hydroxybutyryl-CoA include hbd from C. acetobutylicum (Boynton et al.,J. Bacteriol. 178:3015-3024 (1996), hbd from C. beijerinckii (Colby andChen, Appl. Environ. Microbiol. 58:3297-3302 (1992), and a number ofsimilar enzymes from Metallosphaera sedula (Berg et al., Science318:1782-1786 (2007).

Protein GenBank ID GI Number Organism hbd NP_349314.1 15895965Clostridium acetobutylicum hbd AAM14586.1 20162442 Clostridiumbeijerinckii Msed_1423 YP_001191505 146304189 Metallosphaera sedulaMsed_0399 YP_001190500 146303184 Metallosphaera sedula Msed_0389YP_001190490 146303174 Metallosphaera sedula Msed_1993 YP_001192057146304741 Metallosphaera sedula

The gene product of crt from C. acetobutylicum catalyzes the dehydrationof 3-hydroxybutyryl-CoA to crotonyl-CoA (Boynton et al., J. Bacteriol.178:3015-3024 (1996); Atsumi et al., Metab. Eng. (2007)). Further,enoyl-CoA hydratases are reversible enzymes and thus suitable candidatesfor catalyzing the dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA.The enoyl-CoA hydratases, phaA and phaB, of P. putida are believed tocarry out the hydroxylation of double bonds during phenylacetatecatabolism (Olivera et al., Proc. Nat. Acad. Sci. U.S.A. 95:6419-6424(1998)). The paaA and paaB from P. fluorescens catalyze analogoustransformations (Olivera et al., supra). Lastly, a number of Escherichiacoli genes have been shown to demonstrate enoyl-CoA hydratasefunctionality including maoC, paaF, and paaG (Park and Lee, J.Bacteriol. 185:5391-5397 (2003); Park and Lee, Appl. Biochem.Biotechnol. 113-116:335-346 (2004); Park and Yup, Biotechnol. Bioeng.86:681-686 (2004); Ismail et al., J. Bacteriol. 175:5097-5105 (2003)).

Protein GenBank ID GI Number Organism crt NP_349318.1 15895969Clostridium acetobutylicum paaA NP_745427.1 26990002 Pseudomonas putidapaaB NP_745426.1 26990001 Pseudomonas putida phaA ABF82233.1 106636093Pseudomonas fluorescens phaB ABF82234.1 106636094 Pseudomonasfluorescens maoC NP_415905.1 16129348 Escherichia coli paaF NP_415911.116129354 Escherichia coli paaG NP_415912.1 16129355 Escherichia coli

Several enzymes that naturally catalyze the reverse reaction (i.e., thedehydration of 4-hydroxybutyryl-CoA to crotonoyl-CoA) in vivo have beenidentified in numerous species. This transformation is required for4-aminobutyrate fermentation by Clostridium aminobutyricum (Scherf andBuckel, Eur. J. Biochem. 215:421-429 (1993) and succinate-ethanolfermentation by Clostridium kluyveri (Scherf et al., Arch. Microbiol.161:239-245 (1994)). The transformation is also a key step in Archaea,for example, Metallosphaera sedula, as part of the3-hydroxypropionate/4-hydroxybutyrate autotrophic carbon dioxideassimilation pathway (Berg et al., Science 318:1782-1786 (2007)). Thispathway requires the hydration of crotonoyl-CoA to form4-hydroxybutyryl-CoA. The reversibility of 4-hydroxybutyryl-CoAdehydratase is well-documented (Muh et al., Biochemistry 35:11710-11718(1996); Friedrich et al., Agnew Chem. Int. Ed. Engl. 47:3254-3257(2008); Muh et al., Eur. J Biochem. 248:380-384 (1997) and theequilibrium constant has been reported to be about 4 on the side ofcrotonoyl-CoA (Scherf and Buckel, Eur. J. Biochem. 215:421-429 (1993).This implies that the downstream 4-hydroxybutyryl-CoA dehydrogenase mustkeep the 4-hydroxybutyryl-CoA concentration low so as to not create athermodynamic bottleneck at crotonyl-CoA. The reverse reaction of4-hydroxybutyryl-CoA dehydratase is crotonyl-CoA hydratase.

Protein GenBank ID GI Number Organism AbfD CAB60035 70910046 Clostridiumaminobutyricum AbfD YP_001396399 153955634 Clostridium kluyveriMsed_1321 YP_001191403 146304087 Metallosphaera sedula Msed_1220YP_001191305 146303989 Metallosphaera sedula

Suitable acetoacetyl-CoA and 4-hydroxybutyryl-CoA transferases areencoded by the gene products of cat1, cat2, and cat3 of Clostridiumkluyveri. These enzymes have been shown to exhibit succinyl-CoA,4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity, respectively(Seedorf et al., Proc. Natl. Acad. Sci. USA 105:2128-2133 (2008);Sohling and Gottschalk, J Bacteriol 178:871-880 (1996)). Similar CoAtransferase activities are also present in Trichomonas vaginalis (vanGrinsven et al., J Biol. Chem. 283:1411-1418 (2008)) and Trypanosomabrucei (Riviere et al., J Biol. Chem. 279:45337-45346 (2004)). Yetanother transferase capable of the desired conversions isbutyryl-CoA:acetoacetate CoA-transferase. Exemplary enzymes can be foundin Fusobacterium nucleatum (Barker et al., J Bacteriol. 152(1):201-7(1982)), Clostridium SB4 (Barker et al., J. Biol. Chem. 253(4):1219-25(1978)), and Clostridium acetobutylicum (Wiesenborn et al., Appl.Environ. Microbiol. 55(2):323-9 (1989)). Although specific genesequences were not provided for butyryl-CoA:acetoacetate CoA-transferasein these references, the genes FN0272 and FN0273 have been annotated asa butyrate-acetoacetate CoA-transferase (Kapatral et al., J. Bact.184(7) 2005-2018 (2002)). Homologs in Fusobacterium nucleatum such asFN1857 and FN1856 also likely have the desired acetoacetyl-CoAtransferase activity. FN1857 and FN1856 are located adjacent to manyother genes involved in lysine fermentation and are thus very likely toencode an acetoacetate:butyrate CoA transferase (Kreimeyer, et al., J.Biol. Chem. 282 (10) 7191-7197 (2007)). Additional candidates fromPorphyrmonas gingivalis and Thermoanaerobacter tengcongensis can beidentified in a similar fashion (Kreimeyer, et al., J. Biol. Chem. 282(10) 7191-7197 (2007)). Information related to these proteins and genesis shown below.

Protein GENBANK ID GI NUMBER ORGANISM Cat1 P38946.1 729048 Clostridiumkluyveri Cat2 P38942.2 1705614 Clostridium kluyveri Cat3 EDK35586.1146349050 Clostridium kluyveri TVAG_395550 XP_001330176 123975034Trichomonas vaginalis G3 Tb11.02.0290 XP_828352 71754875 Trypanosomabrucei FN0272 NP_603179.1 19703617 Fusobacterium nucleatum FN0273NP_603180.1 19703618 Fusobacterium nucleatum FN1857 NP_602657.1 19705162Fusobacterium nucleatum FN1856 NP_602656.1 19705161 Fusobacteriumnucleatum PG1066 NP_905281.1 34540802 Porphyromonas gingivalis W83PG1075 NP_905290.1 34540811 Porphyromonas gingivalis W83 TTE0720NP_622378.1 20807207 Thermoanaerobacter tengcongensis MB4 TTE0721NP_622379.1 20807208 Thermoanaerobacter tengcongensis MB4

An alternative method for removing the CoA moiety from acetoacetyl-CoAor 4-hydroxybutyryl-CoA is to apply a pair of enzymes such as aphosphate-transferring acyltransferase and a kinase to impartacetoacetyl-CoA or 4-hydroxybutyryl-CoA synthetase activity. Exemplarynames for these enzymes includephosphotrans-4-hydroxybutyrylase/4-hydroxybutyrate kinase, which canremove the CoA moiety from 4-hydroxybutyryl-CoA, andphosphotransacetoacetylase/acetoacetate kinase which can remove the CoAmoiety from acetoacetyl-CoA. This general activity enables the nethydrolysis of the CoA-ester of either molecule with the simultaneousgeneration of ATP. For example, the butyrate kinase(buk)/phosphotransbutyrylase (ptb) system from Clostridiumacetobutylicum has been successfully applied to remove the CoA groupfrom 3-hydroxybutyryl-CoA when functioning as part of a pathway for3-hydroxybutyrate synthesis (Tseng et al., Appl. Environ. Microbiol.75(10):3137-3145 (2009)). Specifically, the ptb gene from C.acetobutylicum encodes an enzyme that can convert an acyl-CoA into anacyl-phosphate (Walter et al. Gene 134(1): p. 107-11 (1993)); Huang etal. J Mol Microbiol Biotechnol 2(1): p. 33-38 (2000). Additional ptbgenes can be found in butyrate-producing bacterium L2-50 (Louis et al.J. Bacteriol. 186:2099-2106 (2004)) and Bacillus megaterium (Vazquez etal. Curr. Microbiol 42:345-349 (2001)). Additional exemplaryphosphate-transferring acyltransferases include phosphotransacetylase,encoded by pta. The pta gene from E. coli encodes an enzyme that canconvert acetyl-CoA into acetyl-phosphate, and vice versa (Suzuki, T.Biochim. Biophys. Acta 191:559-569 (1969)). This enzyme can also utilizepropionyl-CoA instead of acetyl-CoA forming propionate in the process(Hesslinger et al. Mol. Microbiol 27:477-492 (1998)). Informationrelated to these proteins and genes is shown below.

Protein GENBANK ID GI NUMBER ORGANISM Pta NP_416800.1 16130232Escherichia coli Ptb NP_349676 15896327 Clostridium acetobutylicum PtbAAR19757.1 38425288 butyrate-producing bacterium L2-50 Ptb CAC07932.110046659 Bacillus megaterium

Exemplary kinases include the E. coli acetate kinase, encoded by ackA(Skarstedt and Silverstein J. Biol. Chem. 251:6775-6783 (1976)), the C.acetobutylicum butyrate kinases, encoded by buk1 and buk2 ((Walter etal. Gene 134(1):107-111 (1993); Huang et al. J Mol Microbiol Biotechnol2(1):33-38 (2000)), and the E. coli gamma-glutamyl kinase, encoded byproB (Smith et al. J. Bacteriol. 157:545-551 (1984)). These enzymesphosphorylate acetate, butyrate, and glutamate, respectively. The ackAgene product from E. coli also phosphorylates propionate (Hesslinger etal. Mol. Microbiol 27:477-492 (1998)). Information related to theseproteins and genes is shown below:

Protein GENBANK ID GI NUMBER ORGANISM AckA NP_416799.1 16130231Escherichia coli Buk1 NP_349675 15896326 Clostridium acetobutylicum Buk2Q97II1 20137415 Clostridium acetobutylicum ProB NP_414777.1 16128228Escherichia coli

Example IV 1,4-Butanediol Synthesis Pathway

This Example shows further enzymes that can be used in a 1,4-butanediolpathway. The genes for acetoacetyl-CoA thiolase, 3-Hydroxybutyryl-CoAdehydrogenase (Hbd), Crotonase (Crt), and Crotonyl-CoA hydratase(4-Budh) are described herein above.

Alcohol-forming 4-hydroxybutyryl-CoA reductase enzymes catalyze the 2reduction steps required to form 1,4-butanediol from4-hydroxybutyryl-CoA. Exemplary 2-step oxidoreductases that convert anacyl-CoA to alcohol include those that transform substrates such asacetyl-CoA to ethanol (e.g., adhE from E. coli (Kessler et al., FEBSLett. 281:59-63 (1991)) and butyryl-CoA to butanol (e.g. adhE2 from C.acetobutylicum (Fontaine et al., J. Bacteriol. 184:821-830 (2002)). TheadhE2 enzyme from C. acetobutylicum was specifically shown in ref(WO/2008/115840 (2008)) to produce BDO from 4-hydroxybutyryl-CoA. Inaddition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhEin Leuconostoc mesenteroides has been shown to oxide the branched chaincompound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J. Gen.Appl. Microbiol. 18:43-55 (1972; Koo et al., Biotechnol. Lett.27:505-510 (2005)).

Protein GenBank ID GI Number Organism adhE NP_415757.1 16129202Escherichia coli adhE2 AAK09379.1 12958626 Clostridium acetobutylicumadhE AAV66076.1 55818563 Leuconostoc mesenteroides

Another exemplary enzyme can convert malonyl-CoA to 3-HP. AnNADPH-dependent enzyme with this activity has characterized inChloroflexus aurantiacus where it participates in the3-hydroxypropionate cycle (Hugler et al., J. Bacteriol. 184:2404-2410(2002); Strauss and Fuchs, Eur. J. Biochem. 215:633-643 (1993)). Thisenzyme, with a mass of 300 kDa, is highly substrate-specific and showslittle sequence similarity to other known oxidoreductases (Hugler etal., J. Bacteriol. 184:2404-2410 (2002)). No enzymes in other organismshave been shown to catalyze this specific reaction; however there isbioinformatic evidence that other organisms may have similar pathways(Klatt et al., Environ. Microbiol. 9:2067-2078 (2007)). Enzymecandidates in other organisms including Roseiflexus castenholzii,Erythrobacter sp. NAP1 and marine gamma proteobacterium HTCC2080 can beinferred by sequence similarity.

Protein GenBank ID GI Number Organism mcr AAS20429.1 42561982Chloroflexus aurantiacus Rcas_2929 YP_001433009.1 156742880 Roseiflexuscastenholzii NAP1_02720 ZP_01039179.1 85708113 Erythrobacter sp. NAP1MGP2080_00535 ZP_01626393.1 119504313 marine gamma proteobacteriumHTCC2080

An alternative route to BDO from 4-hydroxybutyryl-CoA involves firstreducing this compound to 4-hydroxybutanal. Several acyl-CoAdehydrogenases are capable of reducing an acyl-CoA to its correspondingaldehyde. Exemplary genes that encode such enzymes include theAcinetobacter calcoaceticus acr1 encoding a fatty acyl-CoA reductase(Reiser and Somerville, J. Bacteriol. 179:2969-2975 (1997), theAcinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al., Appl.Environ. Microbiol. 68:1192-1195 (2002), and a CoA- and NADP-dependentsuccinate semialdehyde dehydrogenase encoded by the sucD gene inClostridium kluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-880(1996); Sohling and Gottschalk, J. Bacteriol. 178:8710880 (1996)). SucDof P. gingivalis is another succinate semialdehyde dehydrogenase(Takahashi et al., J. Bacteriol. 182:4704-4710 (2000)). These succinatesemialdehyde dehydrogenases were specifically shown in ref(WO/2008/115840 (2008)) to convert 4-hydroxybutyryl-CoA to4-hydroxybutanal as part of a pathway to produce 1,4-butanediol. Theenzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encodedby bphG, is yet another capable enzyme as it has been demonstrated tooxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde,isobutyraldehyde and formaldehyde (Powlowski et al., J. Bacteriol.175:377-385 (1993)).

Protein GenBank ID GI Number Organism acr1 YP_047869.1 50086359Acinetobacter calcoaceticus acr1 AAC45217 1684886 Acinetobacter baylyiacr1 BAB85476.1 18857901 Acinetobacter sp. Strain M-1 sucD P38947.1172046062 Clostridium kluyveri sucD NP_904963.1 34540484 Porphyromonasgingivalis bphG BAA03 892.1 425213 Pseudomonas sp

An additional enzyme type that converts an acyl-CoA to its correspondingaldehyde is malonyl-CoA reductase which transforms malonyl-CoA tomalonic semialdehyde. Malonyl-CoA reductase is a key enzyme inautotrophic carbon fixation via the 3-hydroxypropionate cycle inthermoacidophilic archael bacteria (Berg et al., Science 318:1782-1786(2007); Thauer, Science 318:1732-1733 (2007)). The enzyme utilizes NADPHas a cofactor and has been characterized in Metallosphaera andSulfolobus spp (Alber et al., J. Bacteriol. 188:8551-8559 (2006); Hugleret al., J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded byMsed 0709 in Metallosphaera sedula (Alber et al. Mol. Microbiol.61:297-309 (2006); Berg et al., Science 318:1782-1786 (2007)). A geneencoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned andheterologously expressed in E. coli (Alber et al., J. Bacteriol.188:8551-8559 (2006)). Although the aldehyde dehydrogenase functionalityof these enzymes is similar to the bifunctional dehydrogenase fromChloroflexus aurantiacus, there is little sequence similarity. Bothmalonyl-CoA reductase enzyme candidates have high sequence similarity toaspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reductionand concurrent dephosphorylation of aspartyl-4-phosphate to aspartatesemialdehyde. Additional gene candidates can be found by sequencehomology to proteins in other organisms including Sulfolobussolfataricus and Sulfolobus acidocaldarius. Yet another candidate forCoA-acylating aldehyde dehydrogenase is the ald gene from Clostridiumbeijerinckii (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999). Thisenzyme has been reported to reduce acetyl-CoA and butyryl-CoA to theircorresponding aldehydes. This gene is very similar to eutE that encodesacetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth,Appl. Environ. Microbiol. 65:4973-4980 (1999). These proteins areidentified below.

Protein GenBank ID GI Number Organism Msed_0709 YP_001190808.1 146303492Metallosphaera sedula Mcr NP_378167.1 15922498 Sulfolobus tokodaii asd-2NP_343563.1 15898958 Sulfolobus solfataricus Saci_2370 YP_256941.170608071 Sulfolobus acidocaldarius Ald AAT66436 49473535 Clostridiumbeijerinckii eutE AAA80209 687645 Salmonella typhimurium eutE P774452498347 Escherichia coli

4-Hydroxybutyryl-CoA can also be converted to 4-hydroxybutanal inseveral enzymatic steps, though the intermediate 4-hydroxybutyrate.First, 4-hydroxybutyryl-CoA can be converted to 4-hydroxybutyrate by aCoA transferase, hydrolase or synthetase. Alternately,4-hydroxybutyryl-CoA can be converted to 4-hydroxybutyrate via aphosphonated intermediate by enzymes withphosphotrans-4-hydroxybutyrylase and 4-hydroxybutyrate kinase. Exemplarycandidates for these enzymes are described above.

Subsequent conversion of 4-hydroxybutyrate to 4-hydroxybutanal iscatalyzed by an aryl-aldehyde dehydrogenase, or equivalently acarboxylic acid reductase. Such an enzyme is found in Nocardia iowensis.Carboxylic acid reductase catalyzes the magnesium, ATP andNADPH-dependent reduction of carboxylic acids to their correspondingaldehydes (Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007))and is capable of catalyzing the conversion of 4-hydroxybutyrate to4-hydroxybutanal. This enzyme, encoded by car, was cloned andfunctionally expressed in E. coli (Venkitasubramanian et al., J. Biol.Chem. 282:478-485 (2007)). Expression of the npt gene product improvedactivity of the enzyme via post-transcriptional modification. The nptgene encodes a specific phosphopantetheine transferase (PPTase) thatconverts the inactive apo-enzyme to the active holo-enzyme. The naturalsubstrate of this enzyme is vanillic acid, and the enzyme exhibits broadacceptance of aromatic and aliphatic substrates (Venkitasubramanian etal., in Biocatalysis in the Pharmaceutical and Biotechnology Industires,ed. R. N. Patel, Chapter 15, pp. 425-440, CRC Press LLC, Boca Raton,Fla. (2006)).

Gene name GI Number GenBank ID Organism Car 40796035 AAR91681.1 Nocardiaiowensis (sp. NRRL 5646) Npt 114848891 ABI83656.1 Nocardia iowensis (sp.NRRL 5646)

Additional car and npt genes can be identified based on sequencehomology.

Gene name GI Number GenBank ID Organism fadD9 121638475 YP_978699.1Mycobacterium bovis BCG BCG_2812c 121638674 YP_978898.1 Mycobacteriumbovis BCG nfa20150 54023983 YP_118225.1 Nocardia farcinica IFM 10152nfa40540 54026024 YP_120266.1 Nocardia farcinica IFM 10152 SGR_6790182440583 YP_001828302.1 Streptomyces griseus subsp. griseus NBRC 13350SGR_665 182434458 YP_001822177.1 Streptomyces griseus subsp. griseusNBRC 13350 MSMEG_2956 YP_887275.1 YP_887275.1 Mycobacterium smegmatisMC2 155 MSMEG_5739 YP_889972.1 118469671 Mycobacterium smegmatis MC2 155MSMEG_2648 YP_886985.1 118471293 Mycobacterium smegmatis MC2 155MAP1040c NP_959974.1 41407138 Mycobacterium avium subsp.paratuberculosis K-10 MAP2899c NP_961833.1 41408997 Mycobacterium aviumsubsp. paratuberculosis K-10 MMAR_2117 YP_001850422.1 183982131Mycobacterium marinum M MMAR_2936 YP_001851230.1 183982939 Mycobacteriummarinum M MMAR_1916 YP_001850220.1 183981929 Mycobacterium marinum MTpauDRAFT_33060 ZP_04027864.1 227980601 Tsukamurella paurometabola DSM20162 TpauDRAFT_20920 ZP_04026660.1 ZP_04026660.1 Tsukamurellapaurometabola DSM 20162 CPCC7001_1320 ZP_05045132.1 254431429 CyanobiumPCC7001 DDBDRAFT_0187729 XP_636931.1 66806417 Dictyostelium discoideumAX4

An additional enzyme candidate found in Streptomyces griseus is encodedby the griC and griD genes. This enzyme is believed to convert3-amino-4-hydroxybenzoic acid to 3-amino-4-hydroxybenzaldehyde asdeletion of either griC or griD led to accumulation of extracellular3-acetylamino-4-hydroxybenzoic acid, a shunt product of3-amino-4-hydroxybenzoic acid metabolism (Suzuki, et al., J. Antibiot.60(6):380-387 (2007)). Co-expression of griC and griD with SGR_665, anenzyme similar in sequence to the Nocardia iowensis npt, can bebeneficial.

Gene name GI Number GenBank ID Organism griC 182438036 YP_001825755.1Streptomyces griseus subsp. griseus NBRC 13350 Grid 182438037YP_001825756.1 Streptomyces griseus subsp. griseus NBRC 13350

An enzyme with similar characteristics, alpha-aminoadipate reductase(AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in somefungal species. This enzyme naturally reduces alpha-aminoadipate toalpha-aminoadipate semialdehyde. The carboxyl group is first activatedthrough the ATP-dependent formation of an adenylate that is then reducedby NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizesmagnesium and requires activation by a PPTase. Enzyme candidates for AARand its corresponding PPTase are found in Saccharomyces cerevisiae(Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al.,Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe(Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S. pombeexhibited significant activity when expressed in E. coli (Guo et al.,Yeast 21:1279-1288 (2004)). The AAR from Penicillium chrysogenum acceptsS-carboxymethyl-L-cysteine as an alternate substrate, but did not reactwith adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J.Biol. Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenumPPTase has not been identified to date.

Gene name GI Number GenBank ID Organism LYS2 171867 AAA34747.1Saccharomyces cerevisiae LYS5 1708896 P50113.1 Saccharomyces cerevisiaeLYS2 2853226 AAC02241.1 Candida albicans LYS5 28136195 AAO26020.1Candida albicans Lys1p 13124791 P40976.3 Schizosaccharomyces pombe Lys7p1723561 Q10474.1 Schizosaccharomyces pombe Lys2 3282044 CAA74300.1Penicillium chrysogenum

Enzymes exhibiting 1,4-butanediol dehydrogenase activity are capable offorming 1,4-butanediol from 4-hydroxybutanal. Exemplary genes encodingenzymes that catalyze the conversion of an aldehyde to alcohol (i.e.,alcohol dehydrogenase or equivalently aldehyde reductase) include alrAencoding a medium-chain alcohol dehydrogenase for C2-C14 (Tani Appl.Environ. Micro et al. 66:5231-5235 (2000), ADH2 from Saccharomycescerevisiae (Aoshima et al., Mol. Microbiol. 51:791-798 (2004)), yqhDfrom E. coli which has preference for molecules longer than C(3)(Sulzenbacher et al., J. Mol. Biol. 342:489-502 (2004), and bdh I andbdh II from C. acetobutylicum which converts butyraldehyde into butanol(Walter et al., J. Bacteriol. 174:7149-7158 (1992)). ADH1 from Zymomonasmobilis has been demonstrated to have activity on a number of aldehydesincluding formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde,and acrolein (Kinoshita, Appl. Microbiol. Biotechnol. 22:249-254(1985)).

Protein GenBank ID GI Number Organism alrA BAB12273.1 9967138Acinetobacter sp. Strain M-1 ADH2 NP_014032.1 6323961 Saccharymycescerevisiae yqhD NP_417484.1 16130909 Escherichia coli bdh I NP_349892.115896543 Clostridium acetobutylicum bdh II NP_349891.1 15896542Clostridium acetobutylicum adhA YP_162971.1 56552132 Zymomonas mobilis

Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity (EC1.1.1.61) also fall into this category. Such enzymes have beencharacterized in Ralstonia eutropha (Bravo et al., J. Forensic Sci.49:379-387 (2004), Clostridium kluyveri (Wolff and Kenealy, ProteinExpr. Purif. 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz etal., J. Biol. Chem. 278:41552-41556 (2003)).

Protein GenBank ID GI Number Organism 4hbd YP_726053.1 113867564Ralstonia eutropha H16 4hbd L21902.1 146348486 Clostridium kluyveri DSM555 4hbd Q94B07 75249805 Arabidopsis thaliana

Example V Methods for Handling CO and Anaerobic Cultures

This example describes methods used in handling CO and anaerobiccultures.

A. Handling of CO in Small Quantities for Assays and Small Cultures.

CO is an odorless, colorless and tasteless gas that is a poison.Therefore, cultures and assays that utilized CO required specialhandling. Several assays, including CO oxidation, acetyl-CoA synthesis,CO concentration using myoglobin, and CO tolerance/utilization in smallbatch cultures, called for small quantities of the CO gas that weredispensed and handled within a fume hood. Biochemical assays called forsaturating very small quantities (<2 mL) of the biochemical assay mediumor buffer with CO and then performing the assay. All of the CO handlingsteps were performed in a fume hood with the sash set at the properheight and blower turned on; CO was dispensed from a compressed gascylinder and the regulator connected to a Schlenk line. The latterensures that equal concentrations of CO were dispensed to each ofseveral possible cuvettes or vials. The Schlenk line was set upcontaining an oxygen scrubber on the input side and an oil pressurerelease bubbler and vent on the other side. Assay cuvettes were bothanaerobic and CO-containing. Threfore, the assay cuvettes were tightlysealed with a rubber stopper and reagents were added or removed usinggas-tight needles and syringes. Secondly, small (˜50 mL) cultures weregrown with saturating CO in tightly stoppered serum bottles. As with thebiochemical assays, the CO-saturated microbial cultures wereequilibrated in the fume hood using the Schlenk line setup. Both thebiochemical assays and microbial cultures were in portable, sealedcontainers and in small volumes making for safe handling outside of thefume hood. The compressed CO tank was adjacent to the fume hood.

Typically, a Schlenk line was used to dispense CO to cuvettes, eachvented. Rubber stoppers on the cuvettes were pierced with 19 or 20 gagedisposable syringe needles and were vented with the same. An oil bubblerwas used with a CO tank and oxygen scrubber. The glass or quartzspectrophotometer cuvettes have a circular hole on top into which aKontes stopper sleeve, Sz7 774250-0007 was fitted. The CO detector unitwas positioned proximal to the fume hood.

B. Handling of CO in Larger Quantities Fed to Large-Scale Cultures.

Fermentation cultures are fed either CO or a mixture of CO and H₂ tosimulate syngas as a feedstock in fermentative production. Therefore,quantities of cells ranging from 1 liter to several liters can includethe addition of CO gas to increase the dissolved concentration of CO inthe medium. In these circumstances, fairly large and continuouslyadministered quantities of CO gas are added to the cultures. Atdifferent points, the cultures are harvested or samples removed.Alternatively, cells are harvested with an integrated continuous flowcentrifuge that is part of the fermenter.

The fermentative processes are carried out under anaerobic conditions.In some cases, it is uneconomical to pump oxygen or air into fermentersto ensure adequate oxygen saturation to provide a respiratoryenvironment. In addition, the reducing power generated during anaerobicfermentation may be needed in product formation rather than respiration.Furthermore, many of the enzymes for various pathways areoxygen-sensitive to varying degrees. Classic acetogens such as M.thermoacetica are obligate anaerobes and the enzymes in theWood-Ljungdahl pathway are highly sensitive to irreversible inactivationby molecular oxygen. While there are oxygen-tolerant acetogens, therepertoire of enzymes in the Wood-Ljungdahl pathway might beincompatible in the presence of oxygen because most are metallo-enzymes,key components are ferredoxins, and regulation can divert metabolismaway from the Wood-Ljungdahl pathway to maximize energy acquisition. Atthe same time, cells in culture act as oxygen scavengers that moderatethe need for extreme measures in the presence of large cell growth.

C. Anaerobic Chamber and Conditions.

Exemplary anaerobic chambers are available commercially (see, forexample, Vacuum Atmospheres Company, Hawthorne Calif.; MBraun,Newburyport Mass.). Conditions included an O₂ concentration of 1 ppm orless and 1 atm pure N₂. In one example, 3 oxygen scrubbers/catalystregenerators were used, and the chamber included an O₂ electrode (suchas Teledyne; City of Industry Calif.). Nearly all items and reagentswere cycled four times in the airlock of the chamber prior to openingthe inner chamber door. Reagents with a volume >5 mL were sparged withpure N₂ prior to introduction into the chamber. Gloves are changedtwice/yr and the catalyst containers were regenerated periodically whenthe chamber displays increasingly sluggish response to changes in oxygenlevels. The chamber's pressure was controlled through one-way valvesactivated by solenoids. This feature allowed setting the chamberpressure at a level higher than the surroundings to allow transfer ofvery small tubes through the purge valve.

The anaerobic chambers achieved levels of O₂ that were consistently verylow and were needed for highly oxygen sensitive anaerobic conditions.However, growth and handling of cells does not usually require suchprecautions. In an alternative anaerobic chamber configuration, platinumor palladium can be used as a catalyst that requires some hydrogen gasin the mix. Instead of using solenoid valves, pressure release can becontrolled by a bubbler. Instead of using instrument-based O₂monitoring, test strips can be used instead.

D. Anaerobic Microbiology.

Small cultures were handled as described above for CO handling. Inparticular, serum or media bottles are fitted with thick rubber stoppersand aluminum crimps are employed to seal the bottle. Medium, such asTerrific Broth, is made in a conventional manner and dispensed to anappropriately sized serum bottle. The bottles are sparged with nitrogenfor ˜30 min of moderate bubbling. This removes most of the oxygen fromthe medium and, after this step, each bottle is capped with a rubberstopper (such as Bellco 20 mm septum stoppers; Bellco, Vineland, N.J.)and crimp-sealed (Bellco 20 mm). Then the bottles of medium areautoclaved using a slow (liquid) exhaust cycle. At least sometimes aneedle can be poked through the stopper to provide exhaust duringautoclaving; the needle needs to be removed immediately upon removalfrom the autoclave. The sterile medium has the remaining mediumcomponents, for example buffer or antibiotics, added via syringe andneedle. Prior to addition of reducing agents, the bottles areequilibrated for 30-60 minutes with nitrogen (or CO depending upon use).A reducing agent such as a 100×150 mM sodium sulfide, 200 mMcysteine-HCl is added. This is made by weighing the sodium sulfide intoa dry beaker and the cysteine into a serum bottle, bringing both intothe anaerobic chamber, dissolving the sodium sulfide into anaerobicwater, then adding this to the cysteine in the serum bottle. The bottleis stoppered immediately as the sodium sulfide solution generateshydrogen sulfide gas upon contact with the cysteine. When injecting intothe culture, a syringe filter is used to sterilize the solution. Othercomponents are added through syringe needles, such as B12 (10 μMcyanocobalamin), nickel chloride (NiCl₂, 20 microM final concentrationfrom a 40 mM stock made in anaerobic water in the chamber and sterilizedby autoclaving or by using a syringe filter upon injection into theculture), and ferrous ammonium sulfate (final concentration needed is100 μL—made as 100-1000×stock solution in anaerobic water in the chamberand sterilized by autoclaving or by using a syringe filter uponinjection into the culture). To facilitate faster growth under anaerobicconditions, the 1 liter bottles were inoculated with 50 mL of apreculture grown anaerobically. Induction of the pA1-lacO1 promoter inthe vectors was performed by addition of isopropylβ-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mMand was carried out for about 3 hrs.

Large cultures can be grown in larger bottles using continuous gasaddition while bubbling. A rubber stopper with a metal bubbler is placedin the bottle after medium addition and sparged with nitrogen for 30minutes or more prior to setting up the rest of the bottle. Each bottleis put together such that a sterile filter will sterilize the gasbubbled in and the hoses on the bottles are compressible with small Cclamps. Medium and cells are stirred with magnetic stir bars. Once allmedium components and cells are added, the bottles are incubated in anincubator in room air but with continuous nitrogen sparging into thebottles.

Example VI CO Oxidation (CODH) Assay

This example describes assay methods for measuring CO oxidation (COdehydrogenase; CODH).

The 7 gene CODH/ACS operon of Moorella thermoacetica was cloned into E.coli expression vectors. The intact ˜10 kbp DNA fragment was cloned, andit is likely that some of the genes in this region are expressed fromtheir own endogenous promoters and all contain endogenous ribosomalbinding sites. These clones were assayed for CO oxidation, using anassay that quantitatively measures CODH activity. Antisera to the M.thermoacetica gene products was used for Western blots to estimatespecific activity. M. thermoacetica is Gram positive, and ribosomebinding site elements are expected to work well in E. coli. Thisactivity, described below in more detail, was estimated to be ˜ 1/50thof the M. thermoacetica specific activity. It is possible that CODHactivity of recombinant E. coli cells could be limited by the fact thatM. thermoacetica enzymes have temperature optima around 55° C.Therefore, a mesophilic CODH/ACS pathway could be advantageous such asthe close relative of Moorella that is mesophilic and does have anapparently intact CODH/ACS operon and a Wood-Ljungdahl pathway,Desulfitobacterium hafniense. Acetogens as potential host organismsinclude, but are not limited to, Rhodospirillum rubrum, Moorellathermoacetica and Desulfitobacterium hafniense.

CO oxidation is both the most sensitive and most robust of the CODH/ACSassays. It is likely that an E. coli-based syngas using system willultimately need to be about as anaerobic as Clostridial (i.e., Moorella)systems, especially for maximal activity. Improvement in CODH should bepossible but will ultimately be limited by the solubility of CO gas inwater.

Initially, each of the genes was cloned individually into expressionvectors. Combined expression units for multiple subunits/1 complex weregenerated. Expression in E. coli at the protein level was determined.Both combined M. thermoacetica CODH/ACS operons and individualexpression clones were made.

CO oxidation assay. This assay is one of the simpler, reliable, and moreversatile assays of enzymatic activities within the Wood-Ljungdahlpathway and tests CODH (Seravalli et al., Biochemistry 43:3944-3955(2004)). A typical activity of M. thermoacetica CODH specific activityis 500 U at 55° C. or ˜60 U at 25° C. This assay employs reduction ofmethyl viologen in the presence of CO. This is measured at 578 nm instoppered, anaerobic, glass cuvettes.

In more detail, glass rubber stoppered cuvettes were prepared afterfirst washing the cuvette four times in deionized water and one timewith acetone. A small amount of vacuum grease was smeared on the top ofthe rubber gasket. The cuvette was gassed with CO, dried 10 min with a22 Ga. needle plus an exhaust needle. A volume of 0.98 mL of reactionbuffer (50 mM Hepes, pH 8.5, 2 mM dithiothreitol (DTT) was added using a22 Ga. needle, with exhaust needled, and 100% CO. Methyl viologen (CH₃viologen) stock was 1 M in water. Each assay used 20 microliters for 20mM final concentration. When methyl viologen was added, an 18 Ga needle(partial) was used as a jacket to facilitate use of a Hamilton syringeto withdraw the CH₃ viologen. 4-5 aliquots were drawn up and discardedto wash and gas equilibrate the syringe. A small amount of sodiumdithionite (0.1 M stock) was added when making up the CH₃ viologen stockto slightly reduce the CH₃ viologen. The temperature was equilibrated to55° C. in a heated Olis spectrophotometer (Bogart Ga.). A blank reaction(CH₃ viologen+ buffer) was run first to measure the base rate of CH₃viologen reduction. Crude E. coli cell extracts of ACS90 and ACS91(CODH-ACS operon of M. thermoacetica with and without, respectively, thefirst cooC). 10 microliters of extract were added at a time, mixed andassayed. Reduced CH₃ viologen turns purple. The results of an assay areshown in Table I.

TABLE I ACS90 7.7 mg/ml ACS91 11.8 mg/ml Mta98 9.8 mg/ml Mta99 11.2mg/ml Extract Vol OD/ U/ml U/mg ACS90 10 microliters 0.073 0.376 0.049ACS91 10 microliters 0.096 0.494 0.042 Mta99 10 microliters 0.0031 0.0160.0014 ACS90 10 microliters 0.099 0.51 0.066 Mta99 25 microliters 0.0120.025 0.0022 ACS91 25 microliters 0.215 0.443 0.037 Mta98 25 microliters0.019 0.039 0.004 ACS91 10 microliters 0.129 0.66 0.056 Averages ACS900.057 U/mg ACS91 0.045 U/mg Mta99 0.0018 U/mg 

Mta98/Mta99 are E. coli MG1655 strains that express methanolmethyltransferase genes from M. thermoacetia and, therefore, arenegative controls for the ACS90 ACS91 E. coli strains that contain M.thermoacetica CODH operons.

If ˜1% of the cellular protein is CODH, then these figures would beapproximately 100× less than the 500 U/mg activity of pure M.thermoacetica CODH. Actual estimates based on Western blots are 0.5% ofthe cellular protein, so the activity is about 50× less than for M.thermoacetica CODH. Nevertheless, this experiment demonstrates COoxidation activity in recombinant E. coli with a much smaller amount inthe negative controls. The small amount of CO oxidation (CH₃ viologenreduction) seen in the negative controls indicates that E. coli may havea limited ability to reduce CH₃ viologen.

To estimate the final concentrations of CODH and Mtr proteins, SDS-PAGEfollowed by Western blot analyses were performed on the same cellextracts used in the CO oxidation, ACS, methyltransferase, and corrinoidFe—S assays. The antisera used were polyclonal to purified M.thermoacetica CODH-ACS and Mtr proteins and were visualized using analkaline phosphatase-linked goat-anti-rabbit secondary antibody. TheWesterns were performed and results are shown in FIG. 9. The amounts ofCODH in ACS90 and ACS91 were estimated at 50 ng by comparison to thecontrol lanes. Expression of CODH-ACS operon genes including 2 CODHsubunits and the methyltransferase were confirmed via Western blotanalysis. Therefore, the recombinant E. coli cells express multiplecomponents of a 7 gene operon. In addition, both the methyltransferaseand corrinoid iron sulfur protein were active in the same recombinant E.coli cells. These proteins are part of the same operon cloned into thesame cells.

The CO oxidation assays were repeated using extracts of Moorellathermoacetica cells for the positive controls. Though CODH activity inE. coli ACS90 and ACS91 was measurable, it was at about 130-150× lowerthan the M. thermoacetica control. The results of the assay are shown inFIG. 10. Briefly, cells (M. thermoacetica or E. coli with the CODH/ACSoperon; ACS90 or ACS91 or empty vector: pZA33S) were grown and extractsprepared as described above. Assays were performed as described above at55° C. at various times on the day the extracts were prepared. Reductionof methylviologen was followed at 578 nm over a 120 sec time course.

These results describe the CO oxidation (CODH) assay and results.Recombinant E. coli cells expressed CO oxidation activity as measured bythe methyl viologen reduction assay.

Example VII Acetyl-CoA Synthase (ACS) Activity Assay (CO Exchange Assay)

This Example describes an ACS assay method.

This assay measures the ACS-catalyzed exchange of the carbonyl group ofacetyl-CoA with CO (Raybuck et al., Biochemistry 27:7698-7702 (1988)).ACS (as either a purified enzyme or part of a cell extract) is incubatedwith acetyl-CoA labeled with ¹⁴C at the carbonyl carbon under a COatmosphere. In the presence of active ACS, the radioactivity in theliquid phase of the reaction decreases exponentially until it reaches aminimum defined by the equilibrium between the levels of ¹⁴C-labeledacetyl-CoA and ¹⁴C-labeled CO. The same cell extracts of E. coli MG1655expressing ACS90 and ACS91 employed in the other assays as well ascontrol extracts were assayed by this method.

Briefly in more detail, in small assay vials under normal atmosphere, asolution of 0.2 mM acetyl-CoA, 0.1 mM methyl viologen, and 2 mMTi(III)citrate in 0.3M MES buffer, pH 6.0, was made. The total reactionvolume when all components were added was 500 μl. Vials were sealed withrubber stoppers (Bellco) and crimp aluminum seals (Bellco) to create agas-tight reaction atmosphere. Each vial was sparged with 100% CO forseveral minutes, long enough to completely exchange the vials'atmosphere, and brought into an anaerobic chamber. The assay vials wereplaced in a 55° C. sand bath and allowed to equilibrate to thattemperature. A total of 10 scintillation vials with 40 μl of 1M HCl wereprepared for each assay vial. A gas-tight Hamilton syringe was used toadd ACS to the assay vial and incubated for approximately 2-3 minutesfor the reaction to come to equilibrium. A gas-tight Hamilton syringewas used to add 1 μl (0.36 nmoles) ¹⁴C-acetyl-CoA to start the assay(time=0 min). Time points were taken starting immediately. Samples (40μl) were removed from the assay vials with a gas-tight Hamilton syringe.Each sample was added to the 40 μl of HCl in the prepared scintillationvials to quench the reaction. As the ACS enzyme transfers ¹⁴C label toCO from acetyl-CoA, the concentration of the isotope decreasesexponentially. Therefore, the assay was sampled frequently in the earlytime points. The precise time for each sample was recorded. The exactpace of the reaction depends on the ACS enzyme, but generally severalsamples are taken immediately and sampled over the initial 10-15minutes. Samples are continued to be taken for 1-2 hours.

In a particular exemplary assay, four assay conditions were used: blank(no ACS), 12 μl of purified E. coli strains expressing M. thermoaceticaACS, 4 μl of purified E. coli ACS, and 3.7 μl of M. thermoaceticaCODH/ACS. In another exemplary assay, four assay conditions were used:108 μg CODH/ACS, 1 mg Mta99 cell extract, 1 mg ACS90 cell extract, and 1mg ACS91 cell extract. The enzymes were added as 100 μl solutions (50 mMKPi, 0.1M NaCl, pH7.6). A more sensitive assay that can be used for mostof the CODH-ACS activities is the synthesis assay described below. Thisexample describes the assay conditions for measuring ACS activity.

Example VIII Acetyl-CoA Synthesis and Methyltransferase Assays

This example describes acetyl-CoA synthesis and methyltransferaseassays.

Acetyl-CoA synthesis assay. This assay is an in vitro reaction thatsynthesizes acetyl-CoA from methyl-tetrahydrofolate, CO, and CoA usingCODH/ACS, methyltransferase (MeTr), and corrinoid Fe—S protein (CFeSP)(Raybuck et al., Biochemistry 27:7698-7702 (1988)). By adding or leavingout each of the enzymes involved, this assay can be used for a widerange of experiments, from testing one or more purified enzymes or cellextracts for activity, to determining the kinetics of the reaction undervarious conditions or with limiting amounts of substrate or enzyme.Samples of the reaction taken at various time points are quenched with1M HCl, which liberates acetate from the acetyl-CoA end product. Afterpurification with Dowex columns, the acetate can be analyzed bychromatography, mass spectrometry, or by measuring radioactivity. Theexact method can be determined by the specific substrates used in thereaction.

A ¹⁴C-labeled methyl-THF was utilized, and the radioactivity of theisolated acetate samples was measured. The primary purpose was to testCFeSP subunits. The assay also included +/−purified methyltransferaseenzymes. The following 6 different conditions were assayed: (1) purifiedCODH/ACS, MeTr, and CFeSP as a positive control; (2) purified CODH/ACSwith ACS90 cell extract; (3) purified CODH/ACS with ACS91 cell extract;(4) purified CODH/ACS, MeTr with ACS90 cell extract; (5) purifiedCODH/ACS, MeTr with ACS91 cell extract; (6) purified CODH/ACS, MeTr withas much ACS91 cell extract as possible (excluding the MES buffer).

The reaction is assembled in the anaerobic chamber in assay vials thatare filled with CO. The total reaction volume is small compared to thevial volume, so the reagents can be added before or after the vial isfilled with CO, so long as a gas-tight Hamilton syringe is used and thereagents are kept anaerobic. The reaction (˜60 ul total) consisted ofthe cell extract (except assay #1), CoA, Ti(III)citrate, MES (exceptassay #6), purified CODH/ACS, ¹⁴C-methyl-tetrahydrofolate,methyl-viologen, and ferredoxin. Additionally, purified MeTr was addedto assays #1 and #4-6, and purified CFeSP was added to assay #1.

The reaction was carried out in an anaerobic chamber in a sand bath at55° C. The final reagent added was the ¹⁴C-methyl-tetrahydrofolate,which started the reaction (t=Os). An initial sample was takenimmediately, followed by samples at 30 minutes, 1 hour, and 2 hours.These time points are not exact, as the 6 conditions were runconcurrently (since this experiment was primarily a qualitative one).The 15 μl samples were added to 15 μl of 1M HCl in scintillation vials.For the last sample, if less than 15 μl was left in the reactions, theassay vials were rinsed with the 15 ul of HCl to take the remainder ofthe reaction. A volume of 10 μl of cell extract was used for assay #2-5,and 26.4 μl of cell extract was used for assay #6.

Typical amounts of purified enzyme used in the assays is as follows:CODH/ACS=˜0.2 nmoles; MeTr=0.2 nmoles; CFeSP=0.05 nmoles. Typical assayconcentrations are used as follows: CODH/ACS=1 uM; Me-CFeSP=0.4 uM;MeTr=1 uM; Ferredoxin=3 uM; CoA=0.26 mM; ¹⁴C methyl-THF=0.4 mM; methylviologen=0.1 mM; and Ti(III)citrate=3 mM.

After counting the reaction mixtures, it was determined that thecorrinoid Fe—S protein in ACS90 extracts was active with total activityapproaching approximately ⅕ of the positive control and significantlyabove the negative control (no extract).

A non-radioactive synthesis assay can also be used. Optionalnon-radioactive assay conditions are as follows: Assay condition #1: 100mM MES, pH6.0; 1 mM CoA; 1 mM Me-THF; 0.33 mM Ti(III) citrate, volume to950 ul, +50 ul of extract; incubated under a CO atmosphere (Ar forcontrol), at 55° C. These reactions should be carried out in the dark,as the corrinoid methyl carrier is light sensitive. Assay condition #2:100 mM MES, pH6.0; 1 mM CoA; 1 mM Me-THF; 1 mM methyl viologen; volumeto 950 ul, +50 ul of extract; incubated under a CO atmosphere, at 55°C., in the dark. The reaction was quenched with 10 μl of 10% formicacid, with samples taken at 1 hr, 3 hrs, and 6.5 hrs, and stored at˜20°. Assay condition #3: 100 mM Tris, pH 7.6; 5 mM CoA; 7.5 mM Me-THF;1 mM Me-viologen; volume to 90 μl, +10 μl extract; incubated under CO orAr, at 55° C. in the dark for 1 hr, quenched with 10 μl 10% formic acid,and stored at −20° C.

In Lu et al., (J. Biol. Chem. 265:3124-3133. (1990)), the pH optimum forthe synthesis reaction was found to be between 7.2-7.5. Lu et al. alsofound that CoA concentrations above 10 mM were inhibitory. Lu et al.described using methyl iodide as the methyl donor instead of Me-THF, andused 5-7.5 mM concentrations. Lu et al. also determined that DTT orother reducing agents were not necessary, although they did useferredoxin as an electron carrier. Methyl viologen was substituted inthe above-described reactions. In addition, Maynard et al., J. Biol.Inorg. Chem. 9:316-322 (2004), has determined that the electron carrierwas not strictly necessary, but that failure to include one resulted ina time lag of the synthesis. Maynard et al. used 1 mM methyl viologen aselectron carrier when one was used.

Methyltransferase Assay. Within the CODH-ACS operon is encoded anessential methyltransferase activity that catalyzes the transfer of CH₃from methyl-tetrahydrofolate to the ACS complex as part of the synthesisof acetyl-CoA. This is the step that the methyl and carbonyl pathwaysjoin together. Within the operon in M. thermoacetica, the Mtr-encodinggene is Moth_1197 and comes after the main CODH and ACS subunits.Therefore, Mtr activity would constitute indirect evidence that the moreproximal genes can be expressed.

Mtr activity was assayed by spectroscopy. Specifically, methylatedCFeSP, with Co(III), has a small absorption peak at ˜450 nm, whilenon-methylated CFeSP, with Co(I), has a large peak at ˜390 nm. Thisspectrum is due to both the cobalt and iron-sulfur cluster chromophores.Additionally, the CFeSP can spontaneously oxidize to Co(II), whichcreates a broad absorption peak at ˜470 nm (Seravalli et al.,Biochemistry 38:5728-5735 (1999)). Recombinant methyltransferase istested using E. coli cell extracts, purified CFeSP from M.thermoacetica, and methyl-tetrahydrofolate. The methylation of thecorrinoid protein is observed as a decrease in the absorption at 390 nmwith a concurrent increase in the absorption at 450 nm, along with theabsence of a dominant peak at 470 nm.

Non-radioactive assays are also being developed using ¹³C-methanol. Thisshould transfer to tetrahydrofolate and create a MTHF of molecularmass+1. Alternatively, the methyltransferase is thought to also work bytransfer of the methanol methyl group to homocysteine to formmethionine. This assay is also useful because methionine+1 mass is morereadily detectable than MTHF+1 or some other possibilities. In additionto using ¹³C, deuterium can also be used as a tracer, both of which canbe measured using mass spectrometry. These tracers can also be used inin vivo labeling studies. Other assay methods can be used to determinevarious intermediates or products including, for example, electronparamagnetic resonance (EPR), Mossbauer spectroscopy, Electron-NuclearDOuble Resonance (ENDOR), infrared, magnetic circular dichroism (MCD),crystallography, X-ray absorption, as well as kinetic methods, includingstopped flow and freeze-quench EPR.

FIG. 3B illustrates how methanol methyltransferase fits into a CODH/ACS(‘syngas’) pathway. Essentially, the methyl group of methanol istransferred via a cobabalamin-dependent process to tetrahydrofolate andthen to the corrinoid-FeS protein of CODH/ACS (also a cobalamin protein)and that, in turn, donates the methyl group to the ACS reaction thatresults in acetate synthesis. The methanol methyltransferase complexconsists of three gene products; two of these, MtaB and MtaC, (Moth_1209and Moth_1208) are adjacent and were readily cloned. The third, MtaA,may be encoded by three different genes (Moth_2100, Moth_2102, andMoth_2346), and it unclear whether all three genes are required orwhether a subset of the three can function. All cloning in E. coli wasperformed using the Lutz-Bujard vectors (Lutz and Bujard, Nucleic AcidsRes. 25:1203-1210 (1997)).

The following assay can be used to determine the activity of MtaB thatencodes a methanol methyltransferase gene product. A positive controlfor the latter can be performed with vanillate o-demethylation.

Methanol Methyltransferase reaction. An exemplary methanolmethyl-transfer reaction has been described previously (Sauer andThauer, Eur. J. Biochem. 249:280-285 (1997); Naidu and Ragsdale, J.Bacteriol. 183:3276-3281 (2001)). The reaction conditions are asfollows: 50 mM MOPS/KOH, pH 7.0; 10 mM MgCl₂; 4 mM Ti(III) citrate; 0.2%dodecylmaltoside (replacing SDS, see Sauer and. Thauer, Eur. J. Biochem.253:698-705 (1998)); 25 μM hydroxycobalamin; 1% MeOH or 1 mM vanillate(depending on the methyl transferase version). These reactions aremeasured by spectrograph readings in the dark at 37° C. or 55° C. Thisassay tests the ability of MtaB or MtvB to transfer the methyl group tocobalamin from methanol or vanillate, respectively.

Example IX E. coli CO Tolerance Experiment and CO Concentration Assay(Myoglobin Assay)

This example describes the tolerance of E. coli for high concentrationsof CO.

To test whether or not E. coli can grow anaerobically in the presence ofsaturating amounts of CO, cultures were set up in 120 ml serum bottleswith 50 ml of Terrific Broth medium (plus reducing solution, NiCl₂,Fe(II)NH₄SO₄, cyanocobalamin, IPTG, and chloramphenicol) as describedabove for anaerobic microbiology in small volumes. One half of thesebottles were equilibrated with nitrogen gas for 30 min. and one half wasequilibrated with CO gas for 30 min. An empty vector (pZA33) was used asa control, and cultures containing the pZA33 empty vector as well asboth ACS90 and ACS91 were tested with both N₂ and CO. All wereinoculated and grown for 36 hrs with shaking (250 rpm) at 37° C. At theend of the 36 hour period, examination of the flasks showed high amountsof growth in all. The bulk of the observed growth occurred overnightwith a long lag.

Given that all cultures appeared to grow well in the presence of CO, thefinal CO concentrations were confirmed. This was performed using anassay of the spectral shift of myoglobin upon exposure to CO. Myoglobinreduced with sodium dithionite has an absorbance peak at 435 nm; thispeak is shifted to 423 nm with CO. Due to the low wavelength and need torecord a whole spectrum from 300 nm on upwards, quartz cuvettes must beused. CO concentration is measured against a standard curve and dependsupon the Henry's Law constant for CO of maximum water solubility=970micromolar at 20° C. and 1 atm.

For the myoglobin test of CO concentration, cuvettes were washed 10×with water, lx with acetone, and then stoppered as with the CODH assay.N₂ was blown into the cuvettes for ˜10 min. A volume of 1 ml ofanaerobic buffer (HEPES, pH 8.0, 2 mM DTT) was added to the blank (notequilibrated with CO) with a Hamilton syringe. A volume of 10 microlitermyoglobin (˜1 mM—can be varied, just need a fairly large amount) and 1microliter dithionite (20 mM stock) were added. A CO standard curve wasmade using CO saturated buffer added at 1 microliter increments. Peakheight and shift was recorded for each increment. The cultures testedwere pZA33/CO, ACS90/CO, and ACS91/CO. each of these was added in 1microliter increments to the same cuvette. Midway through the experimenta second cuvette was set up and used. The results are shown in Table II.

TABLE II Carbon Monoxide Concentrations, 36 hrs. Strain and GrowthConditions Final CO concentration (micromolar) pZA33-CO 930 ACS90-CO 638494 734 883 ave 687 SD 164 ACS91-CO 728 812 760 611 ave. 728 SD 85

The results shown in Table II indicate that the cultures grew whether ornot a strain was cultured in the presence of CO or not. These resultsindicate that E. coli can tolerate exposure to CO under anaerobicconditions and that E. coli cells expressing the CODH-ACS operon canmetabolize some of the CO.

These results demonstrate that E. coli cells, whether expressingCODH/ACS or not, were able to grow in the presence of saturating amountsof CO. Furthermore, these grew equally well as the controls in nitrogenin place of CO. This experiment demonstrated that laboratory strains ofE. coli are insensitive to CO at the levels achievable in a syngasproject performed at normal atmospheric pressure. In addition,preliminary experiments indicated that the recombinant E. coli cellsexpressing CODH/ACS actually consumed some CO, probably by oxidation tocarbon dioxide.

Example X Enhanced Yield of 1,4-Butanediol from Carbohydrates UsingCO/H₂

This example describes the generation of a microbial organism capable ofproducing 1,4-butanediol from carbohydrates (e.g., glucose) at a yieldgreater than 1 mol 1,4-butanediol/mol glucose. Synthesis gas is suppliedas a secondary source of reducing equivalents to compliment thecarbohydrate-based feedstock.

Escherichia coli is used as a target organism to engineer the1,4-butanediol pathway shown in FIG. 6A. E. coli provides a good hostfor generating a non-naturally occurring microorganism capable ofproducing 1,4-butanediol from a mixed feedstock consisting ofcarbohydrates, CO, and/or H₂ . E. coli is amenable to geneticmanipulation and is known to be capable of producing various products,like ethanol, acetic acid, formic acid, lactic acid, and succinic acid,effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce 1,4-butanediol andextract reducing equivalents from CO and H₂, nucleic acids encoding therequisite enzymes are expressed in E. coli and various non-desirablegenes are targeted for deletion using well known molecular biologytechniques (see, for example, Sambrook, supra, 2001; Ausubel, supra,1999). The construction of a host E. coli strain capable of synthesizing1,4-butanediol from succinyl-CoA is described in (Burk et al., WO2008/115840). Targeted gene deletions of lactate dehydrogenase (ldh),alcohol/aldehyde dehydrogenase (adhE), pyruvate formate lyase (pfl),succinate semialdehyde dehydrogenase (sad and gabD) are implemented forenhancing the yield of 1,4-butanediol.

Suitable host backgrounds include AB3 and ECKh-138 as described byVanDien et al., (2009). Genes integrated into the chromosome or expressedvia plasmids to enable 1,4-butanediol production from succinyl-CoAinclude succinyl-CoA reductase (aldehyde forming) [sucD, NP_904963.1,GI: 34540484, Porphyromonas gingivalis W83], 4-hydroxybutyratedehydrogenase [4hbd, NP_904964.1, GI: 34540485, Porphyromonas gingivalisW83], 4-hydr mn oxybutyryl-CoA transferase [cat2, NP_906037.1, GI:34541558, Porphyromonas gingivalis W83], and 4-hydroxybutyryl-CoAreductase (aldehyde forming) [GNM0025B—codon optimized variant of ald,AAT66436, GI: 49473535, Clostridium beijerinckii—described in Van Dienet al., (2009). Endogenous alcohol dehydrogenases can carry out thereduction of 4-hydroxybutyryaldehyde to 1,4-butanediol. This step can beenhanced by overexpressing a native alcohol dehydrogenase such as (yqhD,NP_417484.1, GI: 16130909, Escherichia coli) or a non-native alcoholdehydrogenase such as (adhA, YP_162971.1, GI: 56552132, Zymomonasmobilis). PEP carboxykinase from E. coli [pck, NP_417862.1, GI:16131280], H. influenzae [pckA, P43923.1, GI: 1172573], or anotherorganism are expressed to improve the energetic efficiency of theengineered pathway.

1,4-Butanediol pathway genes are integrated into the chromosome assynthetic operons. This entails targeted integration using RecET-based‘recombineering’ (Angrand et al., Nucleic Acids Res. 27.17:e16 (1999);Muyrers et al., Nucleic Acids Res. 27.6:1555-1557 (1999); Zhang et al.,Nat. Genet. 20.2:123-128 (1998)). A potential issue with RecET-basedintegration of a cassette and removal of a FRT or loxP-boundedselectable marker by FLP or Cre is the production of a recombinationscar at each integration site. While problems caused by this can beminimized by a number of methods, other means that do not leave genomicscars are available. The standard alternative is to introduce thedesired genes using integrative ‘suicide’ plasmids coupled tocounter-selection such as that allowed by the Bacillus sacB gene (Linket al., J. Bacteriol. 179.20:6228-6237 (1997)); in this way, markerlessand scar less insertions at any location in the E. coli chromosome canbe generated.

Carbon monoxide dehydrogenase activity is enabled by cloning thefollowing genes from Clostridium carboxydivorans P7 (ZP_05391756.1, GI:255524806; ZP_05391757.1, GI: 255524807; ZP_05391758.1, GI: 255524808;ZP_05392945, GI: 255526021) into the pZE13 vector (Expressys, Ruelzheim,Germany) under the PA1/lacO promoter. Alternatively, carbon monoxidedehydrogenase activity is enabled by cloning the following genes fromClostridium carboxydivorans P7 (ZP_05392944, GI: 255526020; ZP_05392945,GI: 255526021) into the pZE13 vector (Expressys, Ruelzheim, Germany)under the PA1/lacO promoter. In addition, ferredoxin andNAD(P)H:ferredoxin oxidoreductase activity is enabled by cloning thefollowing genes from Clostridium carboxydivorans P7 (ZP_05392639.1, GI:255525707; ZP_05392638.1, GI: 255525706; ZP_05392636.1, GI: 255525704;ZP_05392958.1, GI: 255526034) or from Helicobacter pylori (NP_207955.1;GI: 15645778; AAD07340.1, GI: 2313367) into the pZA33 vector (Expressys,Ruelzheim, Germany) under the PA1/lacO promoter. Hydrogenase activity isenabled by cloning the following genes from Ralstonia eutropha H₁₆(HoxF, NP_942727.1, GI: 38637753; HoxU, NP_942728.1, GI: 38637754; HoxY,NP_942729.1, GI: 38637755; HoxH, NP_942730.1, GI: 38637756; HoxW,NP_942731.1, GI: 38637757; HoxI, NP_942732.1, GI: 38637758) into a thirdcompatible plasmid, pZS23, under the PA1/lacO promoter. pZS23 isobtained by replacing the ampicillin resistance module of the pZS13vector (Expressys, Ruelzheim, Germany) with a kanamycin resistancemodule by well-known molecular biology techniques.

Cloned genes are verified by PCR and or restriction enzyme mapping todemonstrate construction and insertion into the expression vector. DNAsequencing of the presumptive clones is carried out to confirm theexpected sequences of each gene. Expression of the cloned genes ismonitored using SDS-PAGE of whole cell extracts. To optimize levels ofsoluble vs. pellet (potentially inclusion body origin) protein, theaffect of titration of the promoter on these levels can be examined. Ifno acceptable expression is obtained, higher or lower copy numbervectors or variants in promoter strength are tested. The three sets ofplasmids are transformed into the 1,4-butanediol-producing host strainof E. coli to express the proteins and enzymes required for theextraction of reducing equivalents from H₂ and CO.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). The expression of the1,4-butanediol synthesis and reducing equivalent extraction genes iscorroborated using methods well known in the art for determiningpolypeptide expression or enzymatic activity, including for example,Northern blots, PCR amplification of mRNA, immunoblotting, and the like.Enzymatic activities of the expressed enzymes are confirmed using assaysspecific for the individual activities. The assay for CO dehydrogenaseactivity is one of the simpler, reliable, and more versatile assays ofenzymatic activities within the Wood-Ljungdahl pathway (Ragsdale andWood, J. Biol. Chem. 260:3970-3977 (1985)). It will provide a measure ofthe activity of CODH in recombinant cells. This assay employs reductionof methyl viologen in the presence of CO. This is measured at 578 nm instoppered, anaerobic, glass cuvettes. Some hydrogenase assays useelectron acceptors such as methyl viologen and test the enzymaticactivity relative to inhibitors such as cyanide (Ragsdale and Ljungdahl,Arch. Microbiol. 139:361-365 (1984); Menon and Ragsdale, Biochemistry36:8484-8494 (1996)). Assays for hydrogenase activity that directlymeasure H₂ generation or loss with GC or H₂ electrodes can also beapplied (Der et al., Anal. Biochem. 150:481-486 (1985)). The ability ofthe engineered E. coli strain to produce 1,4-butanediol is confirmedusing HPLC, gas chromatography-mass spectrometry (GCMS), liquidchromatography-mass spectrometry (LCMS), or other suitable analyticalmethods using routine procedures well known in the art.

Microbial strains engineered to have a functional 1,4-butanediolsynthesis and reducing equivalent extraction pathway are furtheraugmented by optimization for efficient utilization of the pathway.Briefly, the engineered strain is assessed to determine whether any ofthe exogenous genes are expressed at a rate limiting level. Expressionis increased for any enzymes expressed at low levels that can limit theflux through the pathway by, for example, introduction of additionalgene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of 1,4-butanediol. One modeling methodis the bilevel optimization approach, OptKnock (Burgard et al.,Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to selectgene knockouts that collectively result in better production of1,4-butanediol. Adaptive evolution also can be used to generate betterproducers of, for example, the succinyl-CoA intermediate of the1,4-butanediol product. Adaptive evolution is performed to improve bothgrowth and production characteristics (Fong and Palsson, Nat. Genet.36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Basedon the results, subsequent rounds of modeling, genetic engineering andadaptive evolution can be applied to the 1,4-butaendiol producer tofurther increase production.

Initial conditions employ strictly anaerobically grown cells providedwith exogenous glucose as a carbon and energy source. Alternatively, orin addition to glucose, nitrate can be added to the fermentation brothto serve as an electron acceptor and initiator of growth. Anaerobicgrowth of E. coli on fatty acids, which are ultimately metabolized toacetyl-CoA, has been demonstrated in the presence of nitrate (Campbellet al., Mol. Microbiol. 47:793-805 (2003). Oxygen can also be providedas long as its intracellular levels are maintained below any inhibitionthreshold of the engineered enzymes. Anaerobic conditions are maintainedby first sparging the medium with nitrogen, then CO and H₂, and finallysealing the culture vessel, for example, flasks can be sealed with aseptum and crimp-cap. Microaerobic conditions also can be utilized byproviding a small hole in the septum for limited aeration. The pH of themedium is maintained at a pH of around 7 by addition of an acid, such asH₂SO₄. The growth rate is determined by measuring optical density usinga spectrophotometer (600 nm) and the glucose uptake rate by monitoringcarbon source depletion over time. The final product and intermediates,and other organic compounds, can be analyzed by methods such as HPLC(High Performance Liquid Chromatography), GC-MS (Gas Chromatography-MassSpectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) orother suitable analytical methods using routine procedures well known inthe art. The release of product in the fermentation broth can also betested with the culture supernatant. Byproducts and residual glucose canbe quantified by HPLC using, for example, a refractive index detectorfor glucose and alcohols, and a UV detector for organic acids (Lin etal., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay anddetection methods well known in the art.

For large-scale production of 1,4-butanediol, the above organism iscultured in a fermenter using a medium known in the art to supportgrowth of the organism under anaerobic or microaerobic conditions.Fermentations are performed in either a batch, fed-batch or continuousmanner. Previous studies have shown that a rate-limiting step to syngasutilization can be the mass transfer of CO into the liquid phase (Do etal., Biotechnol. Bioeng. 97:279-286 (2007)). This is largely due to therelatively low solubility of CO in water. Continuously gas-spargedfermentations (i.e., with H₂ and CO) are performed in controlledfermenters with continuous off-gas analysis by mass spectrometry andperiodic liquid sampling and analysis by GC and HPLC. 1,4-butanediolproduction, as well as detailed metabolite production, are quantifiedvia GCMS or LCMS. All piping in these systems is glass or metal tomaintain anaerobic conditions. The gas sparging is done using glassfrits to decrease bubble size, maximize the surface to volume ratio, andimprove mass transfer. Various sparging rates are tested, ranging fromabout 0.1 to 1 vvm (vapor volumes per minute). Agitation and impellerdesign are examined. We will also test methods such as moderateoverpressure at 1.5 atm to improve mass transfer (Najafpour and Younesi,38(1-2):223-228 (2006)). To obtain accurate measurements of gas uptakerates, periodic challenges are performed in which the gas flow istemporarily stopped, and the gas phase composition is monitored as afunction of time. We also will evaluate other high mass transfer systemssuch as bubble columns, which allow hydrostatic overpressure and longerbubble retention times. Fermentation conditions are optimized to furtherimprove the productivity and titer of the 1,4-butanediol producingstrains. The data generated from fermentations are analyzed to discernthe impact of each parameter on cell density and alcohol yield. The pHof the culture impacts the growth of the host and may also affect thebioavailability of some of the trace elements. Other parameters to beoptimized include temperature, trace metals innoculum size, ionicstrength and duration of fermentation.

Example XI Engineering the Reverse TCA Cycle into anIsopropanol-Producing Organism

This example describes the generation of a microbial organism that hasbeen engineered to produce isopropanol from glucose and CO₂. Theorganism contains a functional reverse TCA cycle and a pathway forproducing isopropanol from acetyl-CoA as shown in FIG. 2b . Engineeringa functional pathway to produce isopropanol from glucose and CO₂ at highyields will require the optimized expression of a combination ofexogenous and/or endogenous genes.

Escherichia coli is used as a target organism for engineering anisopropanol-producing pathway that utilizes enzymes from the reduced TCAcycle to assimilate CO₂ . E. coli provides a good host for generating anon-naturally occurring microorganism capable of producing isopropanol.E. coli is amenable to genetic manipulation and is known to be capableof producing various products, like ethanol, acetic acid, formic acid,lactic acid, and succinic acid, effectively under anaerobic ormicroaerobic conditions. Engineering a functional pathway to produceisopropanol from glucose and CO₂ at high yields will require theoptimized expression of a combination of exogenous and/or endogenousgenes.

To generate an E. coli strain engineered to produce isopropanol, nucleicacids encoding the enzymes utilized in the isopropanol pathway fromacetyl-CoA are expressed in E. coli using well known molecular biologytechniques (see, for example, Sambrook, supra, 2001; Ausubel supra,1999; Roberts et al., supra, 1989). In particular, an E. coli strain isengineered to produce isopropanol from acetyl-CoA via the route outlinedin Figure XX. Conversion of acetyl-CoA to acetoacetyl-CoA is catalyzedby acetoacetyl-CoA thiolase, an enzyme native to E. coli encoded by atoB(NP_416728). For the first stage of pathway construction, genes encodingenzymes to transform acetoacetyl-CoA to isopropanol are assembled onto avector. In particular, the genes ctfAB (NP_149326.1 and NP_149327.1),adc (NP_149328.1), and adh (AAA23199.2) encodingacetoacetyl-CoA-transferase, acetoacetate decarboxylase and isopropanoldehydrogenase, respectively, are cloned into the pZE13 vector(Expressys, Ruelzheim, Germany), under the control of the PA1/lacOpromoter. The vector is transformed into E. coli strain MG1655 toexpress the proteins and enzymes required for isopropanol synthesis fromacetoacetyl-CoA.

The resulting genetically engineered organism is cultured in glucosecontaining medium following procedures well known in the art (see, forexample, Sambrook et al., supra, 2001). The expression of isopropanolpathway genes is corroborated using methods well known in the art fordetermining polypeptide expression or enzymatic activity, including forexample, Northern blots, PCR amplification of mRNA and immunoblotting.Enzymatic activities of the expressed enzymes are confirmed using assaysspecific for the individually activities. The ability of the engineeredE. coli strain to produce isopropanol through this pathway is confirmedusing HPLC, gas chromatography-mass spectrometry (GCMS) or liquidchromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional isopropanol synthesispathway are further augmented by optimization for efficient utilizationof the pathway. Briefly, the engineered strain is assessed to determinewhether any of the exogenous genes are expressed at a rate limitinglevel. Expression is increased for any enzymes expressed at low levelsthat can limit the flux through the pathway by, for example,introduction of additional gene copy numbers. After successfuldemonstration of enhanced isopropanol production via the activities ofthe exogenous enzymes, the genes encoding these enzymes are insertedinto the chromosome of a wild type E. coli host using methods known inthe art. Such methods include, for example, sequential single crossover(Gay et al., J. Bacteriol. 153:1424-1431 (1983) and Red/ET methods fromGeneBridges (Zhang et al. (2001). Chromosomal insertion provides severaladvantages over a plasmid-based system, including greater stability andthe ability to co-localize expression of pathway genes.

The isopropanol-overproducing host strain is further engineered toassimilate CO₂ via the reductive TCA cycle as shown in FIG. 2B. Many ofthe native E. coli enzymes are capable of operating in the reductivedirection including aconitase, isocitrate dehydrogenase, succinyl-CoAsynthetase, fumarate reductase, fumarase and malate dehydrogenase. Togenerate a strain with a functional RTCA cycle, nucleic acids encodingalpha-ketoglutarate synthase, pyruvate:ferredoxin oxidoreductase,ATP-citrate synthase, ferredoxin and ferredoxin:NADP⁺ reductase areexpressed in the isopropanol-producing host using well known molecularbiology techniques (see, for example, Sambrook, supra, 2001; Ausubelsupra, 1999; Roberts et al., supra, 1989).

In particular, the korAB (BAB21494 and BAB21495), por (YP_428946.1)genes encoding the alpha-ketoglutarate synthase and pyruvate:ferredoxinoxidoreductase, respectively, are cloned into the pZE13 vector(Expressys, Ruelzheim, Germany), under the control of the PA1/lacOpromoter. This plasmid is then transformed into a host strain containinglacI^(Q), which allows inducible expression by addition ofisopropyl-beta-D-1-thiogalactopyranoside (IPTG). The genes aclAB(AAM72321.1 and AAM72322.1), fdx1 (BAE02673.1) and HP1164 (NP_207955.1)encoding ATP-citrate synthase, ferredoxin and ferredoxin:NADP⁺reductase, respectively, are cloned into the pZA33 vector (Expressys,Ruelzheim, Germany) under the PA1/lacO promoter. The two sets ofplasmids are transformed into the isopropanol-producing E. coli strain,described above, to express the proteins and enzymes required for CO₂assimilation to acetyl-CoA, and subsequently isopropanol, via thereductive TCA cycle.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). The expression ofreductive TCA cycle genes is corroborated using methods well known inthe art for determining polypeptide expression or enzymatic activity,including for example, Northern blots, PCR amplification of mRNA andimmunoblotting. Enzymatic activities of the expressed enzymes areconfirmed using assays specific for the individually activities. Theability of the engineered E. coli strain to assimilate CO₂ through thispathway is confirmed using ^(D)C labeled bicarbonate, HPLC, gaschromatography-mass spectrometry (GCMS) or liquid chromatography-massspectrometry (LCMS).

Microbial strains engineered to have a functional reductive TCA pathwayare further augmented by optimization for efficient utilization of thepathway. Briefly, the engineered strain is assessed to determine whetherany of the exogenous genes are expressed at a rate limiting level.Expression is increased for any enzymes expressed at low levels that canlimit the flux through the pathway by, for example, introduction ofadditional gene copy numbers.

After successful demonstration of enhanced isopropanol production viathe activities of the exogenous enzymes, the genes encoding theseenzymes are inserted into the chromosome of a wild type E. coli hostusing methods known in the art. Such methods include, for example,sequential single crossover ((Gay et al., J. Bacteriol. 153:1424-1431(1983) and Red/ET methods from GeneBridges (Zhang et al. (2001).Chromosomal insertion provides several advantages over a plasmid-basedsystem, including greater stability and the ability to co-localizeexpression of pathway genes.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and in U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of isopropanol. One modeling method isthe bilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of isopropanol.Adaptive evolution also can be used to generate better producers of, forexample, acetyl-CoA or acetoacetyl-CoA intermediates or the isopropanolproduct. Adaptive evolution is performed to improve both growth andproduction characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058(2004); Alper et al., Science 314:1565-1568 (2006)). Based on theresults, subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the isopropanol producer to further increaseproduction.

For large-scale production of isopropanol, the above organism iscultured in a fermenter using a medium known in the art to supportgrowth of the organism under anaerobic conditions. Fermentations areperformed in either a batch, fed-batch or continuous manner. Anaerobicconditions are maintained by first sparging the medium with nitrogen andthen sealing culture vessel (e.g., flasks can be sealed with a septumand crimp-cap). Microaerobic conditions also can be utilized byproviding a small hole for limited aeration. The pH of the medium ismaintained at a pH of 7 by addition of an acid, such as H₂SO₄. Thegrowth rate is determined by measuring optical density using aspectrophotometer (600 nm), and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu) with an HPX-087 column (BioRad), using a refractive indexdetector for glucose and alcohols, and a UV detector for organic acids,Lin et al., Biotechnol. Bioeng., 775-779 (2005).

Example XII Introducing the Syngas Utilization Pathway into anIsopropanol Producing Organism

This example describes the generation of a microbial organism that hasbeen engineered to produce isopropanol from glucose and CO₂. Theorganism contains a functional Wood Ljungdahl pathway for fixing carbonas shown in FIG. 4A and a pathway for producing isopropanol fromacetyl-CoA as shown in FIG. 1B.

Escherichia coli is used as a target organism for engineering anisopropanol-producing pathway that utilizes enzymes from the Woodljungdahl pathway to fix CO₂ . E. coli provides a good host forgenerating a non-naturally occurring microorganism capable of producingisopropanol. E. coli is amenable to genetic manipulation and is known tobe capable of producing various products, like ethanol, acetic acid,formic acid, lactic acid, and succinic acid, effectively under anaerobicor microaerobic conditions. Engineering a functional pathway to produceisopropanol from glucose and CO₂ at high yields will require theoptimized expression of a combination of exogenous and/or endogenousgenes.

To generate an E. coli strain engineered to produce isopropanol, nucleicacids encoding the enzymes utilized in the isopropanol pathway fromacetyl-CoA are expressed in E. coli using well known molecular biologytechniques (see, for example, Sambrook, supra, 2001; Ausubel supra,1999; Roberts et al., supra, 1989). In particular, an E. coli strain isengineered to produce isopropanol from acetyl-CoA via the route outlinedin FIG. 1B. Conversion of acetyl-CoA to acetoacetyl-CoA is catalyzed byacetoacetyl-CoA thiolase, an enzyme native to E. coli encoded by atoB(NP_416728). For the first stage of pathway construction, genes encodingenzymes to transform acetoacetyl-CoA to isopropanol are assembled onto avector. In particular, the genes ctfAB (NP_149326.1 and NP_149327.1),adc (NP_149328.1), and adh (AAA23199.2) encodingacetoacetyl-CoA-transferase, acetoacetate decarboxylase and isopropanoldehydrogenase, respectively, are cloned into the pZE13 vector(Expressys, Ruelzheim, Germany), under the control of the PA1/lacOpromoter. The vector is transformed into E. coli strain MG1655 toexpress the proteins and enzymes required for isopropanol synthesis fromacetoacetyl-CoA.

The resulting genetically engineered organism is cultured in glucosecontaining medium following procedures well known in the art (see, forexample, Sambrook et al., supra, 2001). The expression of isopropanolpathway genes is corroborated using methods well known in the art fordetermining polypeptide expression or enzymatic activity, including forexample, Northern blots, PCR amplification of mRNA and immunoblotting.Enzymatic activities of the expressed enzymes are confirmed using assaysspecific for the individually activities. The ability of the engineeredE. coli strain to produce isopropanol through this pathway is confirmedusing HPLC, gas chromatography-mass spectrometry (GCMS) or liquidchromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional isopropanol synthesispathway are further augmented by optimization for efficient utilizationof the pathway. Briefly, the engineered strain is assessed to determinewhether any of the exogenous genes are expressed at a rate limitinglevel. Expression is increased for any enzymes expressed at low levelsthat can limit the flux through the pathway by, for example,introduction of additional gene copy numbers. After successfuldemonstration of enhanced isopropanol production via the activities ofthe exogenous enzymes, the genes encoding these enzymes are insertedinto the chromosome of a wild type E. coli host using methods known inthe art. Such methods include, for example, sequential single crossover(Gay et al., J. Bacteriol. 153:1424-1431 (1983) and Red/ET methods fromGeneBridges (Zhang et al. (2001). Chromosomal insertion provides severaladvantages over a plasmid-based system, including greater stability andthe ability to co-localize expression of pathway genes.

The isopropanol-overproducing host strain is further engineered toassimilate carbon via the Wood Ljungdahl pathway as shown in FIG. 4A.The enzymes required to be active are formate dehydrogenase,Formyltetrahydrofolate synthetase, Methenyltetrahydrofolatecyclohydrolase, Methylenetetrahydrofolate dehydrogenase,Methylenetetrahydrofolate reductase, Methyltetrahydrofolate:corrinoidprotein methyltransferase (AcsE), Corrinoid iron-sulfur protein (AcsD),Nickel-protein assembly protein (AcsF & CooC), Ferredoxin (Orf7),Acetyl-CoA synthase (AcsB & AcsC), Carbon monoxide dehydrogenase (AcsA),Pyruvate formate lyase (Pfl), and Pyruvate ferredoxin oxidoreductase(Por) or pyruvate dehydrogenase (PDH).

While E. coli naturally possesses the capability for some of therequired transformations in the methyl branch (i.e.,methenyltetrahydrofolate cyclohydrolase, methylenetetrahydrofolatedehydrogenase, methylenetetrahydrofolate reductase), it is thought thatthe methyl branch enzymes from acetogens may have significantly higher(50-100×) specific activities than those from non-acetogens (Morton etal., Genetics and Molecular Biology of Anaerobic Bacteria, M. Sebald,Ed., New York: Springer Verlag pp. 389-406 (1992)). Formatedehydrogenase also appears to be specialized for anaerobic conditions(Ljungdahl and Andreesen, FEBS Lett. 54:279-282 (1975)). Therefore,various non-native versions of each of these are expressed in the strainof E. coli capable of methanol and syngas utilization. For example,Moth_2312 and Moth_2314 (Accession numbers YP_431142 and YP_431144respectively) encoding the alpha and beta subunits of formatedehydrogenase, Moth_0109 (GenBank No: YP_428991.1) encoding forformyltetrahydrofolate synthetase, Moth_1516 (Accession no: YP_430368.1)encoding for methenyltetrahydrofolate cyclohydrolase andmethylenetetrahydrofolate dehydrogenase, and Moth_1191 (Accession no:YP_430048.1) encoding for methylenetetrahydrofolate reductase will becloned and combined into an expression vector designed to express themas a set. Initially, a high or medium copy number vector will be chosen(using ColE1 or P15A replicons). The first promoter to be tested is astrongly constitutive promoter such as lambda pL or an IPTG-inducibleversion of this, pL-lacO (Lutz and Bujard, Nucleic Acids Res.25:1203-1210 (1997)). To make an artificial operon, one 5′ terminalpromoter is placed upstream of the set of genes and each gene receives aconsensus rbs element. The order of genes is based on the natural orderwhenever possible. Ultimately, the genes are integrated into the E. colichromosome. Enzyme assays are performed as described in (Ljungdahl andAndreesen, supra; Yamamoto et al., J. Biol. Chem. 258:1826-1832 (1983);Lovell et al., Arch. Microbiol. 149:280-285 (1988); de Mata andRabinowitz, J. Biol. Chem. 255:2569-2577(1980); D'Ari and Rabinowitz, J.Biol. Chem. 266:25953-23958 (1991); Clark and Ljungdahl, J. Biol. Chem.259:10845-10849 (1984); Clark and Ljungdahl, Methods Enzymol.122:392-399 (1986)).

Expression of acetyl CoA synthase/CO dehydrogenase in a foreign hostrequires introducing many, if not all, of the following proteins andtheir corresponding activities: Methyltetrahydrofolate:corrinoid proteinmethyltransferase (AcsE), Corrinoid iron-sulfur protein (AcsD),Nickel-protein assembly protein (AcsF), Ferredoxin (Orf7), Acetyl-CoAsynthase (AcsB and AcsC), Carbon monoxide dehydrogenase (AcsA), andNickel-protein assembly protein (CooC).

The genes required for carbon-monoxide dehydrogenase/acetyl-CoA synthaseactivity typically reside in a limited region of the native genome thatmay be an extended operon (Ragsdale, Crit. Rev. Biochem. Mol. Biol.39:165-195 (2004); Morton et al., J. Biol. Chem. 266:23824-23828 (1991);Roberts et al., Proc. Nat. Acad. Sci. U.S.A. 86:32-36 (1989)). Each ofthe genes in this operon from the acetogen, M. thermoacetica, hasalready been cloned and expressed actively in E. coli (Morton et al., J.Biol. Chem. 266:23824-23828 (1991); Roberts et al., supra; Lu et al., J.Biol. Chem. 268:5605-5614 (1993)). The protein sequences of these genescan be identified by the following GenBank accession numbers.

Protein GenBank ID Organism AcsE YP_430054 Moorella thermoacetica AcsDYP_430055 Moorella thermoacetica AcsF YP_430056 Moorella thermoaceticaOrf7 YP_430057 Moorella thermoacetica AcsC YP_430058 Moorellathermoacetica AcsB YP_430059 Moorella thermoacetica AcsA YP_430060Moorella thermoacetica CooC YP_430061 Moorella thermoacetica

Using standard PCR methods, the entire ACS/CODH operons are assembledinto low or medium copy number vectors such as pZA33-S(P15A-based) orpZS13-S(pSC101-based). The structures and sequences of the cloned genesare confirmed. Expression is monitored via protein gel electrophoresisof whole-cell lysates grown under strictly anaerobic conditions with therequisite metals (Ni, Zn, Fe) and coenzyme B12 provided. As necessary,the gene cluster is modified for E. coli expression by identificationand removal of any apparent terminators and introduction of consensusribosomal binding sites chosen from sites known to be effective in E.coli (Barrick et al., Nucleic Acids Res. 22:1287-1295 (1994); Ringquistet al., Mol. Microbiol. 6:1219-1229 (1992)). However, each gene clusteris cloned and expressed in a manner parallel to its native structure andexpression. This helps ensure the desired stoichiometry between thevarious gene products—most of which interact with each other.

E. coli possesses native pyruvate formate lyase activity. The resultinggenetically engineered organism is cultured in glucose-containing mediumfollowing procedures well known in the art (see, for example, Sambrooket al., supra, 2001). The expression of the methyl/carbonyl branch genesis corroborated using methods well known in the art for determiningpolypeptide expression or enzymatic activity, including for example,Northern blots, PCR amplification of mRNA and immunoblotting. Enzymaticactivities of the expressed enzymes are confirmed using assays specificfor the individually activities. The ability of the engineered E. colistrain to assimilate carbon through this pathway is confirmed using ¹³Clabeled bicarbonate, HPLC, gas chromatography-mass spectrometry (GCMS)or liquid chromatography-mass spectrometry (LCMS). Initial conditionsemploy strictly anaerobically grown cells provided with exogenousglucose as a carbon and energy source via substrate-levelphosphorylation or anaerobic respiration with nitrate as an electronacceptor. Additionally, exogenously provided CH₃-THF is added to themedium.

Microbial strains engineered to have a functional W-L pathway arefurther augmented by optimization for efficient utilization of thepathway. Briefly, the engineered strain is assessed to determine whetherany of the exogenous genes are expressed at a rate limiting level.Expression is increased for any enzymes expressed at low levels that canlimit the flux through the pathway by, for example, introduction ofadditional gene copy numbers.

After successful demonstration of enhanced isopropanol production viathe activities of the exogenous enzymes, the genes encoding theseenzymes are inserted into the chromosome of a wild type E. coli hostusing methods known in the art. Such methods include, for example,sequential single crossover (Gay et al., J. Bacteriol. 153:1424-1431(1983) and Red/ET methods from GeneBridges (Zhang et al. (2001).Chromosomal insertion provides several advantages over a plasmid-basedsystem, including greater stability and the ability to co-localizeexpression of pathway genes.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and in U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of isopropanol. One modeling method isthe bilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of isopropanol.Adaptive evolution also can be used to generate better producers of, forexample, acetyl-CoA or acetoacetyl-CoA intermediates or the isopropanolproduct. Adaptive evolution is performed to improve both growth andproduction characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058(2004); Alper et al., Science 314:1565-1568 (2006)). Based on theresults, subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the isopropanol producer to further increaseproduction.

For large-scale production of isopropanol, the above organism iscultured in a fermenter using a medium known in the art to supportgrowth of the organism under anaerobic conditions. Fermentations areperformed in either a batch, fed-batch or continuous manner. Anaerobicconditions are maintained by first sparging the medium with nitrogen andthen sealing culture vessel (e.g., flasks can be sealed with a septumand crimp-cap). Microaerobic conditions also can be utilized byproviding a small hole for limited aeration. The pH of the medium ismaintained at a pH of 7 by addition of an acid, such as H₂SO₄. Thegrowth rate is determined by measuring optical density using aspectrophotometer (600 nm), and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu) with an HPX-087 column (BioRad), using a refractive indexdetector for glucose and alcohols, and a UV detector for organic acids,Lin et al., Biotechnol. Bioeng., 775-779 (2005).

Example XIII Engineering the Methanol Utilization Pathway into anIsopropanol Producing Organism

This Example shows how an organism is engineered to utilize methanol inan isopropanol producing organism.

The first step in the cloning and expression process is to express in E.coli the minimal set of genes (e.g., MtaA, MtaB, and MtaC) necessary toproduce Methyl-THF from methanol (FIG. 4B). These methyltransferaseactivities require Coenzyme B12 (cobalamin) as a cofactor. In Moorellathermoacetica, a cascade of methyltransferase proteins mediateincorporation of methanol derived methyl groups into the acetyl-CoAsynthase pathway. Recent work (Das et al., Proteins 67:167-176 (2007)suggests that MtaABC are encoded by Moth_1208-09 and Moth_2346. Thesegenes are cloned via proof-reading PCR and linked together forexpression in a high-copy number vector such as pZE22-S under control ofthe repressible PA1-lacO1 promoter (Lutz and Bujard, Nucleic Acids Res.25:1203-1210 (1997). Cloned genes are verified by PCR and or restrictionenzyme mapping to demonstrate construction and insertion of the 3-geneset into the expression vector. DNA sequencing of the presumptive clonesis carried out to confirm the expected sequences of each gene. Onceconfirmed, the final construct is expressed in E. coli K-12 (MG1655)cells by addition of IPTG inducer between 0.05 and 1 mM finalconcentration. Expression of the cloned genes is monitored usingSDS-PAGE of whole cell extracts. To optimize levels of soluble vs.pellet (potentially inclusion body origin) protein, the affect oftitration of the promoter on these levels can be examined. If noacceptable expression is obtained, higher or lower copy number vectorsor variants in promoter strength are tested.

To determine if expression of the MtaABC proteins from M. thermoaceticaconfers upon E. coli the ability to transfer methyl groups from methanolto tetrahydrofolate (THF) the recombinant strain is fed methanol atvarious concentrations. Activity of the methyltransferase system isassayed anaerobically as described for vanillate as a methyl source inM. thermoacetica (Naidu and Ragsdale, J. Bacteriol. 183:3276-3281 (2001)or for Methanosarcina barkeri methanol methyltransferase (Sauer et al.,Eur. J. Biochem. 243:670-677 (1997); Tallant et al., J. Biol. Chem.276:4485-4493 (2001); Tallant and Krzycki, J. Bacteriol. 179:6902-6911(1997); Tallant and Krzycki, J. Bacteriol. 178:1295-1301 (1996)). For apositive control, M. thermoacetica cells are cultured in parallel andassayed anaerobically to confirm endogenous methyltransferase activity.Demonstration of dependence on exogenously added coenzyme B12 confirmsmethanol:corrinoid methyltransferase activity in E. coli.

Once methyltransferase expression is achieved, further work is performedtowards optimizing the expression. Titrating the promoter in theexpression vector enables the testing of a range of expression levels.This is then used as a guide towards the expression required insingle-copy, or enables the determination of whether or not asingle-copy of these genes allows sufficient expression. If so, themethyltransferase genes are integrated into the chromosome as a single,synthetic operon. This entails targeted integration using RecET-based‘recombineering’ (Angrand et al., Nucleic Acids Res. 27:e16 (1999);Muyrers et al., Nucleic Acids Res. 27:1555-1557 (1999); Zhang et al.,Nat. Genet. 20:123-128 (1998)). A potential issue with RecET-basedintegration of a cassette and removal of a FRT or loxP-boundedselectable marker by FLP or Cre is the production of a recombinationscar at each integration site. While problems caused by this can beminimized by a number of methods, other means that do not leave genomicscars are available. The standard alternative, is to introduce thedesired genes using integrative ‘suicide’ plasmids coupled tocounter-selection such as that allowed by the Bacillus sacB gene (Linket al., J. Bacteriol. 179:6228-6237 (1997); in this way, markerless andscar less insertions at any location in the E. coli chromosome can begenerated. The final goal is a strain of E. coli K-12 expressingmethanol:corrinoid methyltransferase activity under an induciblepromoter and in single copy (chromosomally integrated).

Expression of acetyl CoA synthase/CO dehydrogenase in a foreign hostrequires introducing many, if not all, of the following proteins andtheir corresponding activities. Methyltetrahydrofolate:corrinoid proteinmethyltransferase (AcsE), Corrinoid iron-sulfur protein (AcsD),Nickel-protein assembly protein (AcsF), Ferredoxin (Orf7), Acetyl-CoAsynthase (AcsB and AcsC), Carbon monoxide dehydrogenase (AcsA), andNickel-protein assembly protein (CooC).

The genes required for carbon-monoxide dehydrogenase/acetyl-CoA synthaseactivity typically reside in a limited region of the native genome thatmay be an extended operon (Ragsdale, Crit. Rev. Biochem. Mol. Biol.39:165-195 (2004); Morton et al., J. Biol. Chem. 266:23824-23828 (1991);Roberts et al., Proc. Natl. Acad. Sci. U.S.A. 86:32-36 (1989)). Each ofthe genes in this operon from the acetogen, M. thermoacetica, hasalready been cloned and expressed actively in E. coli (Morton et al., J.Biol. Chem. 266:23824-23828 (1991); Roberts et al., Proc. Natl. Acad.Sci. U.S.A. 86:32-36 (1989); Lu et al., J. Biol. Chem. 268:5605-5614(1993)). The protein sequences of these genes can be identified by thefollowing GenBank accession numbers.

Protein GenBank ID Organism AcsE YP_430054 Moorella thermoacetica AcsDYP_430055 Moorella thermoacetica AcsF YP_430056 Moorella thermoaceticaOrf7 YP_430057 Moorella thermoacetica AcsC YP_430058 Moorellathermoacetica AcsB YP_430059 Moorella thermoacetica AcsA YP_430060Moorella thermoacetica CooC YP_430061 Moorella thermoacetica

Using standard PCR methods, the entire ACS/CODH operons are assembledinto low or medium copy number vectors such as pZA33-S(P15A-based) orpZS13-S(pSC101-based). The structures and sequences of the cloned genesare confirmed. Expression is monitored via protein gel electrophoresisof whole-cell lysates grown under strictly anaerobic conditions with therequisite metals (Ni, Zn, Fe) and coenzyme B12 provided. As necessary,the gene cluster is modified for E. coli expression by identificationand removal of any apparent terminators and introduction of consensusribosomal binding sites chosen from sites known to be effective in E.coli (Barrick et al., Nucleic Acids Res. 22:1287-1295 (1994); Ringquistet al., Mol. Microbiol. 6:1219-1229 (1992)). However, each gene clusteris cloned and expressed in a manner parallel to its native structure andexpression. This helps ensure the desired stoichiometry between thevarious gene products—most of which interact with each other.

E. coli possesses native pyruvate formate lyase activity. The resultinggenetically engineered organism is cultured in glucose-containing mediumfollowing procedures well known in the art (see, for example, Sambrooket al., supra, 2001). The expression of the exogenous genes iscorroborated using methods well known in the art for determiningpolypeptide expression or enzymatic activity, including for example,Northern blots, PCR amplification of mRNA and immunoblotting. Enzymaticactivities of the expressed enzymes are confirmed using assays specificfor the individually activities. The ability of the engineered E. colistrain to assimilate carbon through this pathway is confirmed using ¹³Clabeled bicarbonate, HPLC, gas chromatography-mass spectrometry (GCMS)or liquid chromatography-mass spectrometry (LCMS). Initial conditionsemploy strictly anaerobically grown cells provided with exogenousglucose as a carbon and energy source via substrate-levelphosphorylation or anaerobic respiration with nitrate as an electronacceptor. Additionally, exogenously provided CH₃-THF is added to themedium.

Microbial strains engineered to have a functional methanol utilizationpathway are further augmented by optimization for efficient utilizationof the pathway. Briefly, the engineered strain is assessed to determinewhether any of the exogenous genes are expressed at a rate limitinglevel. Expression is increased for any enzymes expressed at low levelsthat can limit the flux through the pathway by, for example,introduction of additional gene copy numbers.

After successful demonstration of enhanced isopropanol production viathe activities of the exogenous enzymes, the genes encoding theseenzymes are inserted into the chromosome of a wild type E. coli hostusing methods known in the art. Such methods include, for example,sequential single crossover (Gay et al., J. Bacteriol. 153:1424-1431(1983) and Red/ET methods from GeneBridges (Zhang et al. (2001).Chromosomal insertion provides several advantages over a plasmid-basedsystem, including greater stability and the ability to co-localizeexpression of pathway genes.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and in U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of isopropanol. One modeling method isthe bilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of isopropanol.Adaptive evolution also can be used to generate better producers of, forexample, acetyl-CoA or acetoacetyl-CoA intermediates or the isopropanolproduct. Adaptive evolution is performed to improve both growth andproduction characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058(2004); Alper et al., Science 314:1565-1568 (2006)). Based on theresults, subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the isopropanol producer to further increaseproduction.

For large-scale production of isopropanol, the above organism iscultured in a fermenter using a medium known in the art to supportgrowth of the organism under anaerobic conditions. Fermentations areperformed in either a batch, fed-batch or continuous manner. Anaerobicconditions are maintained by first sparging the medium with nitrogen andthen sealing culture vessel (e.g., flasks can be sealed with a septumand crimp-cap). Microaerobic conditions also can be utilized byproviding a small hole for limited aeration. The pH of the medium ismaintained at a pH of 7 by addition of an acid, such as H₂SO₄. Thegrowth rate is determined by measuring optical density using aspectrophotometer (600 nm), and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu) with an HPX-087 column (BioRad), using a refractive indexdetector for glucose and alcohols, and a UV detector for organic acids,Lin et al., Biotechnol. Bioeng., 775-779 (2005).

Example XIV Engineering Cobalamin Synthesis into an Organism

This example describes engineering de novo B12 synthetic capability intoan organism. One enzyme of the Wood-Ljungdahl pathway, ACS/CODH, usescobalamin (vitamin B12) to function. B12 is synthesized de novo in someorganisms but must be supplied exogenously to others. Still otherorganisms such as S. cerevisiae lack the ability to efficiently uptakeB12.

B12 biosynthetic pathways have been characterized in several organismsincluding Salmonella typhimurium LT2 (Roth et al., J. Bacteriol.175:3303-3316 (1993), Lactobacillus reuteri CRL1098 (Santos et al.,Microbiology 154:81-93 (2008) and Bacillus megaterium (Brey et al., J.Bacteriol. 167:623-630 (1986)). Bacterial B12 biosynthesis pathwaysinvolve 20-30 genes clustered together in one or more operons. Twocobalamin biosynthesis pathways: late-insertion (aerobic only) andearly-insertion (anaerobic) have been described (Scott, A. I., J. Org.Chem. 68:2529-2539 (2003)). The final products of the biosynthesis ofvitamin B12 are 5′-deoxyadenosylcobalamin (coenzyme B12) andmethylcobalamin (MeCbl). Vitamin B12 is defined as cyanocobalamin(CNCbl) which is the form commonly prepared in industry. In thisexample, B12 refers to all three analogous molecules.

The anaerobic cobalamin biosynthesis pathway has been well-characterizedin Salmonella typhimurium LT2 (Roth et al., J. Bacteriol. 175:3303-3316(1993)). Pathway genes are clustered in a large operon termed the coboperon. A plasmid containing the following 20 genes from the cob operon(pAR8827) was transformed into E. coli and conferred the ability tosynthesize cobalamin de novo (Raux et al., J. Bacteriol. 178:753-767(1996)). To further improve yield of the cobyric acid precursor, theknown regulatory elements of cbiA were removed and the RBS altered. Thegenes and corresponding GenBank identifiers and gi numbers are listedbelow.

Protein GenBank ID GI Number Organism cysG NP_462380.1 16766765Salmonella typhimurium cbiK NP_460970.1 16765355 Salmonella typhimuriumcbiL NP_460969.1 16765354 Salmonella typhimurium cbiH NP_460972.116765357 Salmonella typhimurium cbiF NP_460974.1 16765359 Salmonellatyphimurium cbiG NP_460973.1 16765358 Salmonella typhimurium cbiDNP_460977.1 16765362 Salmonella typhimurium cbiJ NP_460971.1 16765356Salmonella typhimurium cbiE NP_460976.1 16765361 Salmonella typhimuriumcbiT NP_460975.1 16765360 Salmonella typhimurium cbiC NP_460978.116765363 Salmonella typhimurium cbiA NP_460980.1 16765365 Salmonellatyphimurium fldA NP_459679.1 16764064 Salmonella typhimurium cobAP31570.1 399274 Salmonella typhimurium cbiP AAA27268.1 154436 Salmonellatyphimurium cbiB Q05600.1 543942 Salmonella typhimurium cobU NP_460963.116765348 Salmonella typhimurium cobT NP_460961.1 16765346 Salmonellatyphimurium Cobs AAA27270.1 154438 Salmonella typhimurium cobCNP_459635.1 16764020 Salmonella typhimurium cysG NP_462380.1 16766765Salmonella typhimurium

Some organisms unable to synthesize B12 de novo are able to catalyzesome steps of the pathway. E. coli, for example, is unable to synthesizethe corrin ring structure but encodes proteins that catalyze severalreactions in the pathway (Raux et al., J. Bacteriol. 178:753-767(1996)). The cysG gene encodes a functional CysG, a multifunctionalenzyme that converts uroporphyrinogen III to precorrin-2 (Warren et al.1990; Woodcock et al. 1998). The proteins encoded by cobTSU transformcobinamide to cobalamin and introduce the 5′-deoxyadenosyl group (Rauxet al., J. Bacteriol. 178:753-767 (1996)).

Protein GenBank ID GI Number Organism cobT NP_416495.1 16129932Escherichia coli K12 sp. cobs NP_416496.1 16129933 Escherichia coli K12sp. cobU NP_416497.1 16129934 Escherichia coli K12 sp. cysG NP_417827.116131246 Escherichia coli K12 sp.

S. cerevisiae is not able to synthesize B12 de novo, nor is it able touptake the vitamin at detectable levels. However, the S. cerevisiaegenome encodes two proteins, Metlp and Met8p, that catalyze several B12pathway reactions. Metlp is analogous to the uroporphyrinogen IIItransmethylase CysG of S. typhimurium, which catalyzes the first step ofB12 biosynthesis from uroporphyrinogen III (Raux et al., Biochem. J.338(Pt 3): 701-708 (1999)). The Met8p protein is a bifunctional proteinwith uroporphyrinogen III transmethylase activity and cobaltochelataseactivity analogous to the CysG of B. megaterium (Raux et al., supra(1999)).

Protein GenBank ID GI Number Organism Met1p NP_012995.1 6322922Saccharomyces cerevisiae Met8p NP_009772.1 6319690 Saccharomycescerevisiae

Any or all of these genes can be introduced into an organism deficientor inefficient in one or more components of cobalamin synthesis toenable or increase the efficiency of cobalamin synthesis.

Example XV Engineering Enhanced Cobalamin Uptake Capability in anOrganism

This example describes engineering B12 uptake capability into a hostorganism. B12 uptake requires a specific transport system (Sennett etal., Annu. Rev. Biochem. 50:1053-1086 (1981)).

The B12 transport system of E. coli has been extensively studied.High-affinity transport across the outer membrane is calcium-dependentand mediated by a 66 kDa outer membrane porin, BtuB (Heller et al., J.Bacteriol. 161:896-903 (1985)). BtuB interacts with the TonB energytransducing system (TonB-ExbB-ExbD), facilitating energy-dependenttranslocation and binding to periplasmic binding protein BtuF (Letainand Postle, 1997; Chimento et al. 2003). Transport across the innermembrane is facilitated by an ABC type uptake system composed of BtuF,BtuD (ATP binding component) and BtuC (permease) (Locher et al., Science296:1091-1098 (2002)). Crystal structures of the BtuCDF complex areavailable (Hvorup et al., Science 317:1387-1390 (2007); Locher et al.supra). An additional protein, BtuE, is coexpressed in the btuCEDoperon, but this protein is not required for B12 transport and itsfunction is unknown (Rioux and Kadner, Mol. Gen. Genet. 217:301-308(1989)). The btuCED operon is constitutively expressed. The GenBankidentifiers and GI numbers of the genes associated with B12 transportare listed below.

Protein GenBank ID GI Number Organism btuB NP_418401.1 16131804Escherichia coli K12 sp. MG1655 btuC NP_416226.1 16129667 Escherichiacoli K12 sp. MG1655 btuD NP_416224.1 16129665 Escherichia coli K12 sp.MG1655 btuF NP_414700.1 16128151 Escherichia coli K12 sp. MG1655 tonBNP_415768.1 16129213 Escherichia coli K12 sp. MG1655 exbB NP_417479.116130904 Escherichia coli K12 sp. MG1655 exbD NP_417478.1 16130903Escherichia coli K12 sp. MG1655

The B12 uptake capability of an organism can be further improved byoverexpressing genes encoding the requisite transport proteins, andreducing or eliminating negative regulatory control. Overexpressing thebtuBCDF genes leads to increased binding of B12 to membranes andincreased rate of uptake into cells. Another strategy is to removeregulatory control. The btuB mRNA translation is directly repressed byB12 at the 5′ UTR (Nahvi et al., Chem. Biol. 9:1043 (2002)). Thisinteraction may induce mRNA folding to block ribosome access to thetranslational start. Mutation or elimination of the B12 binding siteremoves inhibition and improves the efficiency of B12 uptake (U.S. Pat.No. 6,432,686 (2002)). These strategies were successfully employed toimprove B12 uptake capability in 1,3-PDO producing microorganisms(WO/1999/058686) and (U.S. Pat. No. 6,432,686 (2002)). A recent patentapplication describes improving the efficiency of B12 uptake(WO/2008/152016) by deleting negative regulatory proteins such as C.glutamicum btuR2.

S. typhimurium possesses both high and low affinity transporters forB12. The high affinity transporter is encoded by btuB (Rioux and Kadner,J. Bacteriol. 171:2986-2993 (1989)). Like E. coli transport across theperiplasmic membrane is predicted to occur via an ABC transport system,although this has not been characterized to date. The B12 bindingprotein is encoded by btuD and btuE, and btuC is predicted to encode thepermease.

Protein GenBank ID GI Number Organism btuB AAA27031.1 153891 Salmonellatyphimurium LT2 btuC NP_460306.1 16764691 Salmonella typhimurium LT2btuD NP_460308.1 16764693 Salmonella typhimurium LT2 btuE AAL20266.116419860 Salmonella typhimurium LT2

Any or all of these genes can be introduced into an organism deficientin one or more components of cobalamin uptake to enable or increase theefficiency cobalamin uptake.

Method for Quantifying B12 in the Culture Medium.

To quantify the amount of B12 in the culture medium, cell free samplesare run on HPLC. Cobalamin quantification is achieved by comparing peakarea ratios at 278 nm and 361 num with standards, then applying peakareas to standard curves of cobalamin.

Throughout this application various publications have been referencedwithin parentheses. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific examples and studies detailed above are onlyillustrative of the invention. It should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. A non-naturally occurring microbial organism having a reductive TCApathway, wherein said microbial organism comprises at least oneexogenous nucleic acid encoding a reductive TCA pathway enzyme expressedin a sufficient amount to enhance carbon flux through acetyl-CoA,wherein said at least one exogenous nucleic acid is selected from anATP-citrate lyase, citrate lyase, a fumarate reductase, and analpha-ketoglutarate:ferredoxin oxidoreductase. 2.-7. (canceled)
 8. Thenon-naturally occurring microbial organism of claim 1 further comprisingan exogenous nucleic acid encoding an enzyme selected from apyruvate:ferredoxin oxidoreductase, an aconitase, an isocitratedehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, afumarase, a malate dehydrogenase, an acetate kinase, aphosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxinoxidoreductase, ferredoxin, and combinations thereof.
 9. Thenon-naturally occurring microbial organism of claim 1 further comprising(a) an isopropanol pathway, said isopropanol pathway convertingacetyl-CoA to isopropanol, wherein said isopropanol pathway comprises 1)an acetoacetyl-CoA thiolase, 2) an acetoacetyl-CoA transferase, anacetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or aphosphotransacetoacetylase/acetoacetate kinase, 3) an acetoacetatedecarboxylase, and 4) an isopropanol dehydrogenase; (b) a 1,3-butanediolpathway; said 1,3-butanediol pathway converting acetyl-CoA to1,3-butanediol, wherein said 1,3-butanediol pathway comprises at leastthree enzymes selected from 1) Acetoacetyl-CoA thiolase (AtoB), 2)Acetoacetyl-CoA reductase (CoA-dependent, alcohol forming), 3)3-oxobutyraldehyde reductase (aldehyde reducing), 4)4-hydroxy,2-butanone reductase, 5) Acetoacetyl-CoA reductase(CoA-dependent, aldehyde forming), 6) 3-oxobutyraldehyde reductase(ketone reducing), 7) 3-hydroxybutyraldehyde reductase, 8)Acetoacetyl-CoA reductase (ketone reducing), 9) 3-hydroxybutyryl-CoAreductase (aldehyde forming), 10) 3-hydroxybutyryl-CoA reductase(alcohol forming), 11) an acetoacetyl-CoA transferase, anacetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or aphosphotransacetoacetylase/acetoacetate kinase, 12) Acetoacetatereductase, 13) 3-hydroxybutyryl-CoA transferase, hydrolase, orsynthetase, 14) 3-hydroxybutyrate reductase, and 15) 3-hydroxybutyratedehydrogenase; (c) a 1,4-butanediol pathway, said 1,4-butanediol pathwayconverting acetyl-CoA to 1,4-butanediol, wherein said 1,4-butanediolpathway comprises at least five enzymes selected from 1) Acetoacetyl-CoAthiolase (AtoB), 2) 3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3)Crotonase (Crt), 4) Crotonyl-CoA hydratase (4-Budh), 5)4-hydroxybutyryl-CoA reductase (alcohol forming), 6)4-hydroxybutyryl-CoA reductase (aldehyde forming), 7) 1,4-butanedioldehydrogenase, 8) 4-Hydroxybutyryl-CoA transferase, 4-Hydroxybutyryl-CoAsynthetase, 4-Hydroxybutyryl-CoA hydrolase, orPhosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate kinase, and 9)4-Hydroxybutyrate reductase; and/or (d) a 4-hydroxybutyrate pathway,said 4-hydroxybutyrate pathway converting acetyl-CoA to4-hydroxybutyrate, wherein said 4-hydroxybutyrate pathway comprises atleast five enzymes selected from 1) Acetoacetyl-CoA thiolase (AtoB), 2)3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3) Crotonase (Crt), 4)Crotonyl-CoA hydratase (4-Budh), 5) 4-Hydroxybutyryl-CoA transferase,hydrolase or synthetase, 6) Phosphotrans-4-hydroxybutyrylase, and 7)4-Hydroxybutyrate kinase. 10.-29. (canceled)
 30. The non-naturallyoccurring microbial organism of claim 1 further comprising an exogenousnucleic acid encoding an enzyme selected from carbon monoxidedehydrogenase, hydrogenase, NAD(P)H:ferredoxin oxidoreductase,ferredoxin, and combinations thereof.
 31. The non-naturally occurringmicrobial organism of claim 30, wherein said microbial organism utilizesa carbon feedstock selected from CO, CO₂, and Hz, synthesis gascomprising CO and H₂, and synthesis gas comprising CO, CO₂, and H₂. 32.A method for enhancing carbon flux through acetyl-CoA, comprisingculturing the non-naturally occurring microbial organism of claim 1under conditions and for a sufficient period of time to produce aproduct having acetyl-CoA as a building block. 33.-62. (canceled)
 63. Anon-naturally occurring microbial organism having a Wood-Ljungdahlpathway, wherein said microbial organism comprises at least oneexogenous nucleic acid encoding a Wood-Ljungdahl pathway enzymeexpressed in a sufficient amount to enhance carbon flux throughacetyl-CoA, wherein said at least one exogenous nucleic acid is selectedfrom a) Formate dehydrogenase, b) Formyltetrahydrofolate synthetase, c)Methenyltetrahydrofolate cyclohydrolase, d) Methylenetetrahydrofolatedehydrogenase, e) Methylenetetrahydrofolate reductase,Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), g)Corrinoid iron-sulfer protein (AcsD), h) Nickel-protein assembly protein(AcsF & CooC), i) Ferredoxin (Orf7), j) Acetyl-CoA synthase (AcsB &AcsC), k) Carbon monoxide dehydrogenase (AcsA), and l) Pyruvateferredoxin oxidoreductase or pyruvate dehydrogenase, and m) pyruvateformate lyase. 64.-76. (canceled)
 77. The non-naturally occurringmicrobial organism of claim 63 further comprising (a) an isopropanolpathway, said isopropanol pathway converting acetyl-CoA to isopropanol,wherein said isopropanol pathway comprises 1) an acetoacetyl-CoAthiolase, 2) an acetoacetyl-CoA transferase, an acetoacetyl-CoAhydrolase, an acetoacetyl-CoA synthetase, or aphosphotransacetoacetylase/acetoacetate kinase, 3) an acetoacetatedecarboxylase, and 4) an isopropanol dehydrogenases; (b) a1,3-butanediol pathway, said 1,3-butanediol pathway convertingacetyl-CoA to 1,3-butanediol, wherein said 1,3-butanediol pathwaycomprises at least three enzymes selected from 1) Acetoacetyl-CoAthiolase (AtoB), 2) Acetoacetyl-CoA reductase (CoA-dependent, alcoholforming), 3) 3-oxobutyraldehyde reductase (aldehyde reducing), 4)4-hydroxy,2-butanone reductase, 5) Acetoacetyl-CoA reductase(CoA-dependent, aldehyde forming), 6) 3-oxobutyraldehyde reductase(ketone reducing), 7) 3-hydroxybutyraldehyde reductase, 8)Acetoacetyl-CoA reductase (ketone reducing), 9) 3-hydroxybutyryl-CoAreductase (aldehyde forming), 10) 3-hydroxybutyryl-CoA reductase(alcohol forming), 11) an acetoacetyl-CoA transferase, anacetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or aphosphotransacetoacetylase/acetoacetate kinase, 12) Acetoacetatereductase, 13) 3-hydroxybutyryl-CoA transferase, hydrolase, orsynthetase, 14) 3-hydroxybutyrate reductase, and 15) 3-hydroxybutyratedehydrogenase, (c) a 1,4-butanediol pathway, said 1,4-butanediol pathwayconverting acetyl-CoA to 1,4-butanediol, wherein said 1,4-butanediolpathway comprises at least five enzymes selected from 1) Acetoacetyl-CoAthiolase (AtoB), 2) 3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3)Crotonase (Crt), 4) Crotonyl-CoA hydratase (4-Budh), 5)4-hydroxybutyryl-CoA reductase (alcohol forming), 6)4-hydroxybutyryl-CoA reductase (aldehyde forming), 7) 1,4-butanedioldehydrogenase, 8) 4-Hydroxybutyryl-CoA transferase, 4-Hydroxybutyryl-CoAsynthetase, 4-Hydroxybutyryl-CoA hydrolase, orPhosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate kinase, and 9)4-Hydroxybutyrate reductase; and/or (d) a 4-hydroxybutyrate pathway,said 4-hydroxybutyrate pathway converting acetyl-CoA to4-hydroxybutyrate, wherein said 4-hydroxybutyrate pathway comprises atleast five enzymes selected from 1) Acetoacetyl-CoA thiolase (AtoB), 2)3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3) Crotonase (Crt), 4)Crotonyl-CoA hydratase (4-Budh), 5) 4-Hydroxybutyryl-CoA transferase,hydrolase or synthetase, 6) Phosphotrans-4-hydroxybutyrylase, and 7)4-Hydroxybutyrate kinase. 78.-97. (canceled)
 98. A method for enhancingcarbon flux through acetyl-CoA, comprising culturing the non-naturallyoccurring microbial organism of claim 63 under conditions and for asufficient period of time to produce a product having acetyl-CoA as abuilding block. 99.-132. (canceled)
 133. A non-naturally occurringmicrobial organism having a methanol Wood-Ljungdahl pathway, whereinsaid microbial organism comprises at least one exogenous nucleic acidencoding a methanol Wood-Ljungdahl pathway enzyme expressed in asufficient amount to enhance carbon flux through acetyl-CoA, whereinsaid at least one exogenous nucleic acid is selected from a) Methanolmethyltransferase (MtaB), b) Corrinoid protein (MtaC), c)Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA), d)Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), e)Corrinoid iron-sulfer protein (AcsD), f) Nickel-protein assembly protein(AcsF & CooC), g) Ferredoxin (Orf7), h) Acetyl-CoA synthase (AcsB &AcsC), i) Carbon monoxide dehydrogenase (AcsA), j) Pyruvate ferredoxinoxidoreductase or pyruvate dehydrogenase, k) pyruvate formate lyase, andl) NAD(P)H:ferredoxin oxidoreductase. 134.-144. (canceled)
 145. Thenon-naturally occurring microbial organism of claim 133 furthercomprising (a) an isopropanol pathway, said isopropanol pathwayconverting acetyl-CoA to isopropanol, wherein said isopropanol pathwaycomprises 1) an acetoacetyl-CoA thiolase, 2) an acetoacetyl-CoAtransferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoAsynthetase, or a phosphotransacetoacetylase/acetoacetate kinase, 3) anacetoacetate decarboxylase, and 4) an isopropanol dehydrogenase; (b) a1,3-butanediol pathway; said 1,3-butanediol pathway convertingacetyl-CoA to 1,3-butanediol, wherein said 1,3-butanediol pathwaycomprises at least three enzymes selected from 1) Acetoacetyl-CoAthiolase (AtoB), 2) Acetoacetyl-CoA reductase (CoA-dependent, alcoholforming), 3) 3-oxobutyraldehyde reductase (aldehyde reducing), 4)4-hydroxy,2-butanone reductase, 5) Acetoacetyl-CoA reductase(CoA-dependent, aldehyde forming), 6) 3-oxobutyraldehyde reductase(ketone reducing), 7) 3-hydroxybutyraldehyde reductase, 8)Acetoacetyl-CoA reductase (ketone reducing), 9) 3-hydroxybutyryl-CoAreductase (aldehyde forming), 10) 3-hydroxybutyryl-CoA reductase(alcohol forming), 11) an acetoacetyl-CoA transferase, anacetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or aphosphotransacetoacetylase/acetoacetate kinase, 12) Acetoacetatereductase, 13) 3-hydroxybutyryl-CoA transferase, hydrolase, orsynthetase, 14) 3-hydroxybutyrate reductase, and 15) 3-hydroxybutyratedehydrogenase; (c) a 1,4-butanediol pathway, said 1,4-butanediol pathwayconverting acetyl-CoA to 1,4-butanediol, wherein said 1,4-butanediolpathway comprises at least five enzymes selected from 1) Acetoacetyl-CoAthiolase (AtoB), 2) 3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3)Crotonase (Crt), 4) Crotonyl-CoA hydratase (4-Budh), 5)4-hydroxybutyryl-CoA reductase (alcohol forming), 6)4-hydroxybutyryl-CoA reductase (aldehyde forming), 7) 1,4-butanedioldehydrogenase, 8) 4-Hydroxybutyryl-CoA transferase, 4-Hydroxybutyryl-CoAsynthetase, 4-Hydroxybutyryl-CoA hydrolase, orPhosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate kinase, and 9)4-Hydroxybutyrate reductase; and/or (d) a 4-hydroxybutyrate pathway,said 4-hydroxybutyrate pathway converting acetyl-CoA to4-hydroxybutyrate, wherein said 4-hydroxybutyrate pathway comprises atleast five enzymes selected from 1) Acetoacetyl-CoA thiolase (AtoB), 2)3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3) Crotonase (Crt), 4)Crotonyl-CoA hydratase (4-Budh), 5) 4-Hydroxybutyryl-CoA transferase,hydrolase or synthetase, 6) Phosphotrans-4-hydroxybutyrylase, and 7)4-Hydroxybutyrate kinase. 146.-165. (canceled)
 166. A method forenhancing carbon flux through acetyl-CoA, comprising culturing thenon-naturally occurring microbial organism of claim 133 under conditionsand for a sufficient period of time to produce a product havingacetyl-CoA as a building block. 167.-199. (canceled)
 200. Anon-naturally occurring microbial organism comprising at least oneexogenous nucleic acid encoding an enzyme expressed in a sufficientamount to enhance the availability of reducing equivalents in thepresence of carbon monoxide or hydrogen, thereby increasing the yield ofredox-limited products via carbohydrate-based carbon feedstock, whereinsaid at least one exogenous nucleic acid is selected from a carbonmonoxide dehydrogenase, a hydrogenase, an NAD(P)H:ferredoxinoxidoreductase, and a ferredoxin. 201.-206. (canceled)
 207. Thenon-naturally occurring microbial organism of claim 200 furthercomprising one or more nucleic acids encoding an enzyme selected from aphosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, apyruvate carboxylase, and a malic enzyme; and/or one or more nucleicacids encoding an enzyme selected from a malate dehydrogenase, afumarase, a fumarate reductase, a succinyl-CoA synthetase, and asuccinyl-CoA transferase.
 208. (canceled)
 209. The non-naturallyoccurring microbial organism of claim 200 further comprising (a) a1,4-butanediol pathway, wherein said microbial organism comprises atleast one exogenous nucleic acid encoding an enzyme selected from 1)Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoAligase), 2) Succinyl-CoA reductase (aldehyde forming), 3)4-Hydroxybutyrate dehydrogenase, 4) 4-Hydroxybutyrate kinase, 5)Phosphotrans-4-hydroxybutyrylase, 6) 4-Hydroxybutyryl-CoA reductase(aldehyde forming), 7) 1,4-butanediol dehydrogenase, 8) Succinatereductase, 9) Succinyl-CoA reductase (alcohol forming), 10)4-Hydroxybutyryl-CoA transferase, 4-Hydroxybutyryl-CoA hydrolase, or4-Hydroxybutyryl-CoA synthetase, 11) 4-Hydroxybutyrate reductase, 12)4-Hydroxybutyryl-phosphate reductase, and 13) 4-Hydroxybutyryl-CoAreductase (alcohol forming); (b) a 1,3-butanediol pathway, wherein saidmicrobial organism comprises at least one exogenous nucleic acidencoding an enzyme selected from 1) Succinyl-CoA transferase, orSuccinyl-CoA synthetase (or succinyl-CoA ligase), 2) Succinyl-CoAreductase (aldehyde forming), 3) 4-Hydroxybutyrate dehydrogenase, 4)4-Hydroxybutyrate kinase, 5) Phosphotrans-4-hydroxybutyrylase, 6)4-Hydroxybutyryl-CoA dehydratase, 7) Crotonase, 8) 3-Hydroxybutyryl-CoAreductase (aldehyde forming), 9) 3-Hydroxybutyraldehyde reductase, 10)Succinate reductase, 11) Succinyl-CoA reductase (alcohol forming), 12)4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase,13) 3-Hydroxybutyryl-CoA reductase (alcohol forming), 14)3-Hydroxybutyryl-CoA hydrolase, or 3-Hydroxybutyryl-CoA synthetase, or3-Hydroxybutyryl-CoA transferase, 15) 3-Hydroxybutyrate reductase;and/or (c) a butanol pathway, wherein said microbial organism comprisesat least one exogenous nucleic acid encoding an enzyme selected from 1)Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoAligase), 2) Succinyl-CoA reductase (aldehyde forming), 3)4-Hydroxybutyrate dehydrogenase, 4) 4-Hydroxybutyrate kinase, 5)Phosphotrans-4-hydroxybutyrylase, 6) 4-Hydroxybutyryl-CoA dehydratase,7) Butyryl-CoA dehydrogenase, 8) Butyryl-CoA reductase (aldehydeforming), 9) Butyraldehyde reductase, 10) Succinate reductase, 11)Succinyl-CoA reductase (alcohol forming), 12) 4-Hydroxybutyryl-CoAtransferase, or 4-Hydroxybutyryl-CoA synthetase, 13) Butyryl-CoAreductase (alcohol forming), 14) Butyryl-CoA hydrolase, or Butyryl-CoAsynthetase, or Butyryl-CoA transferase, 15) Butyrate reductase.210.-216. (canceled)
 217. A method for enhancing the availability ofreducing equivalents in the presence of carbon monoxide or hydrogenthereby increasing the yield of redox-limited products viacarbohydrate-based carbon feedstock, the method comprising culturing thenon-naturally occurring microbial organism of claim 200 under conditionsand for a sufficient period of time to produce a product. 218.-233.(canceled)
 234. A non-naturally occurring microbial organism having: areductive TCA pathway, wherein said microbial organism comprises atleast one exogenous nucleic acid encoding a reductive TCA pathwayenzyme; said at least one exogenous nucleic acid is selected from anATP-citrate lyase, citrate lyase, a fumarate reductase, and analpha-ketoglutarate:ferredoxin oxidoreductase; and at least oneexogenous enzyme selected from a carbon monoxide dehydrogenase, ahydrogenase, a NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin,expressed in a sufficient amount to allow the utilization of 1) CO, 2)CO₂ and H₂, 3) CO and CO₂, 4) synthesis gas comprising CO and H₂, and 5)synthesis gas comprising CO, CO₂, and H₂.
 235. The non-naturallyoccurring microbial organism of claim 234 further comprising at leastone exogenous nucleic acid encoding a citrate lyase, an aconitase, anisocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoAtransferase, a fumarase, a malate dehydrogenase, an acetate kinase, aphosphotransacetylase, and an acetyl-CoA synthetase,
 236. Thenon-naturally occurring microbial organism of claim 234 furthercomprising (a) an isopropanol pathway, said isopropanol pathwayconverting acetyl-CoA to isopropanol, wherein said isopropanol pathwaycomprises 1) an acetoacetyl-CoA thiolase, 2) an acetoacetyl-CoAtransferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoAsynthetase, or a phosphotransacetoacetylase/acetoacetate kinase, 3) anacetoacetate decarboxylase, and 4) an isopropanol dehydrogenase; (b) a1,3-butanediol pathway, said 1,3-butanediol pathway convertingacetyl-CoA to 1,3-butanediol, wherein said 1,3-butanediol pathwaycomprises at least three enzymes selected from 1) Acetoacetyl-CoAthiolase (AtoB), 2) Acetoacetyl-CoA reductase (CoA-dependent, alcoholforming), 3) 3-oxobutyraldehyde reductase (aldehyde reducing), 4)4-hydroxy,2-butanone reductase, 5) Acetoacetyl-CoA reductase(CoA-dependent, aldehyde forming), 6) 3-oxobutyraldehyde reductase(ketone reducing), 7) 3-hydroxybutyraldehyde reductase, 8)Acetoacetyl-CoA reductase (ketone reducing), 9) 3-hydroxybutyryl-CoAreductase (aldehyde forming), 10) 3-hydroxybutyryl-CoA reductase(alcohol forming), 11) an acetoacetyl-CoA transferase, anacetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or aphosphotransacetoacetylase/acetoacetate kinase, 12) Acetoacetatereductase, 13) 3-hydroxybutyryl-CoA transferase, hydrolase, orsynthetase, 14) 3-hydroxybutyrate reductase, and 15) 3-hydroxybutyratedehydrogenase; (c) a 1,4-butanediol pathway, said 1,4-butanediol pathwayconverting acetyl-CoA to 1,4-butanediol, wherein said 1,4-butanediolpathway comprises at least five enzymes selected from 1) Acetoacetyl-CoAthiolase (AtoB), 2) 3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3)Crotonase (Crt), 4) Crotonyl-CoA hydratase (4-Budh), 5)4-hydroxybutyryl-CoA reductase (alcohol forming), 6)4-hydroxybutyryl-CoA reductase (aldehyde forming), 7) 1,4-butanedioldehydrogenase, 8) 4-Hydroxybutyryl-CoA transferase, 4-Hydroxybutyryl-CoAsynthetase, 4-Hydroxybutyryl-CoA hydrolase, orPhosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate kinase, and 9)4-Hydroxybutyrate reductase; and/or (d) a 4-hydroxybutyrate pathway,said 4-hydroxybutyrate pathway converting acetyl-CoA to4-hydroxybutyrate, wherein said 4-hydroxybutyrate pathway comprises atleast five enzymes selected from 1) Acetoacetyl-CoA thiolase (AtoB), 2)3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3) Crotonase (Crt), 4)Crotonyl-CoA hydratase (4-Budh), 5) 4-Hydroxybutyryl-CoA transferase,hydrolase or synthetase, 6) Phosphotrans-4-hydroxybutyrylase, and 7)4-Hydroxybutyrate kinase. 237.-256. (canceled)
 257. A method comprisingculturing the non-naturally occurring microbial organism of claim 234under conditions and for a sufficient period of time to produce aproduct. 258.-279. (canceled)